The level of DNA modification by (+)-syn-(11S,12R,13S,14R)- and ()-anti-(11R,12S,13S,14R)-dihydrodiol epoxides of dibenzo[a,l]pyrene determined the effect on the proteins p53 and p21WAF1 in the human mammary carcinoma cell line MCF-7
Andreas Luch1,2,
Kim Kudla3,
Albrecht Seidel4,
Johannes Doehmer2,
Helmut Greim2,5 and
William M. Baird1,6
1 Departments of Environmental and Molecular Toxicology and Biochemistry and Biophysics, Oregon State University, Agricultural and Life Sciences 1011, Corvallis, OR 97331-7302, USA,
2 Institute of Toxicology and Environmental Hygiene, Technical University of Munich, 80636 Munich, Germany,
3 Biochemistry and Molecular Biology Program, Purdue University, West Lafayette, IN 47907, USA,
4 Institute of Toxicology, University of Mainz, 55131 Mainz, Germany and
5 GSF National Research Center for Environment and Health, Institute of Toxicology, 85764 Neuherberg, Germany
 |
Abstract
|
---|
The polycyclic aromatic hydrocarbon (PAH) dibenzo[a,l]pyrene (DB[a,l]P), the most carcinogenic PAH tested in rodent bioassays, exerts its pathobiological activity via metabolic formation of electrophilically reactive DNAbinding fjord region (+)-syn-(11S,12R,13S,14R)- or ()-anti-(11R,12S,13S,14R)-DB[a,l]P-dihydrodiol epoxides (DB[a,l]PDEs). DB[a,l]P is metabolized to these DB[a,l]PDEs which bind to DNA in human mammary carcinoma MCF-7 cells. The molecular response of MCF-7 cells to DNA damage caused by DB[a,l]PDEs was investigated by analyzing effects on the expression of the tumor suppressor protein p53 and one of its target gene products, the cyclin-dependent kinase inhibitor p21WAF1. Treatment of MCF-7 cells with (+)-syn- and ()-anti-DB[a,l]PDE at a concentration range of 0.0010.1 µM resulted in DB[a,l]PDEDNA adduct levels between 2 and 30, and 3 and 80 pmol/mg DNA, respectively, 8 h after exposure. ()-anti-DB[a,l]PDE exhibited a higher binding efficiency that correlated with a significantly stronger p53 response at low concentrations of the dihydrodiol epoxides. The level of p53 increased by 68 h after treatment. The p21WAF1 protein amount exceeded control levels by 12 h and remained elevated for 96 h. At a dose of 0.01 µM (+)-syn-DB[a,l]PDE, an increase in p21WAF1 was observed in the absence of a detectable change in p53 levels. The results indicate that the increase in p53 induced by DB[a,l]PDEs in MCF-7 cells requires an adduct level of ~15 pmol/mg DNA and suggest that the level of adducts rather than the specific structure of the DB[a,l]PDEDNA adduct formed triggers the p53 response. The PAHDNA adduct level formed may determine whether p53 and p21WAF1 pathways respond, resulting in cell-cycle arrest, or fail to respond and increase the risk of mutation induction by these DNA lesions.
Abbreviations: ()-anti-DB[a,l]PDE, dibenzo[a,l]pyrene-11R,12S-dihydrodiol 13S,14R-epoxide; (+)-anti-B[a]PDE, B[a]P-7R,8S-dihydrodiol 9S,10R-epoxide; (+)-syn-DB[a,l]PDE, dibenzo[a,l]pyrene-11S,12R-dihydrodiol 13S,14R-epoxide; B[a]P, benzo[a]pyrene; CDK, cyclin-dependent kinase; DB[a,l]P, dibenzo[a,l]pyrene; DB[a,l]PDE(s), DB[a,l]P-11,12-dihydrodiol 13,14-epoxide(s); DMSO, dimethyl sulfoxide; FCS, fetal calf serum; PAGE, polyacrylamide gel electrophoresis; PAH(s), polycyclic aromatic hydrocarbon(s); PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate; TBS, Tris-buffered saline; TCPI, trypsinchymotrypsin protease inhibitor.
 |
Introduction
|
---|
The hexacyclic aromatic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P) (Figure 1
) is the most highly tumorigenic polycyclic aromatic hydrocarbon (PAH) tested to date. The tumor-initiating activity of DB[a,l]P exceeds that of benzo[a]pyrene (B[a]P) and even of 7,12-dimethylbenz[a]anthracene (DMBA), formerly thought to be the most potent carcinogenic PAH after application to mouse skin or rat mammary gland (13). DB[a,l]P has been detected as a widespread pollutant in the human environment (48), and several laboratories have investigated the mechanism of DNA damage induction by this compound. Studies in mammalian cell cultures including human cell lines (9,10), in mouse skin in vivo (9), and in microsomal preparations (11) revealed that cytochrome P450 enzymes activate DB[a,l]P to its electrophilically reactive fjord region 11,12-dihydrodiol 13,14-epoxides (DB[a,l]PDEs) (Figure 1
) which predominantly bind to deoxyadenosine residues within DNA. Analysis of the stereochemical course of the metabolic activation in human mammary carcinoma MCF-7 cells (10) demonstrated that this PAH is exclusively converted to (+)-syn-(11S,12R,13S,14R)- and ()-anti-(11R,12S, 13S,14R)-DB[a,l]PDE via their corresponding precursors, the (+)-(11S,12S)- and ()-(11R,12R)-dihydrodiols, respectively (Figure 1
). No formation of (+)-anti-(11S,12R,13R,14S)- and ()-syn-(11R,12S,13R,14S)-DB[a,l]PDE was detected (10). Although only racemic fjord region syn- and anti-DB[a,l]PDEs have been tested, their extraordinarily strong mutagenic activity in Salmonella typhimurium and Chinese hamster V79 cells (12), and their high carcinogenic potency in mouse skin, newborn mouse and rat mammary gland (1315) may account for the high tumorigenicity of DB[a,l]P. Although it has been proposed that DB[a,l]P can also be activated through a radical cation intermediate to produce unstable depurinating DNA adducts (11), no increase in apurinic sites was detected in MCF-7 cells exposed to DB[a,l]P and its 11,12-dihydrodiol 13,14-epoxides (16). The DNA damage induced in MCF-7 cells treated with DB[a,l]P results from the formation of stable covalent DB[a,l]PDEDNA adducts only (16).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 1. Schematic representation of the stereoselective metabolism of dibenzo[a,l]pyrene in human mammary carcinoma MCF-7 cells.
|
|
Covalent modification of genomic DNA by metabolically formed DB[a,l]PDEs (10,11,17,18) represents a type of cellular DNA damage demonstrated previously to be responsible for an increase in the tumor suppressor protein p53. Cells containing wild-type p53 phosphoprotein are able to recognize DNA damage caused not only by metabolites of PAH (1922), but also by UV light (23), ionizing radiation (24) or antitumor drugs (25). The initial cellular response consists of a nuclear accumulation of p53, transcriptional induction of various target genes containing p53-binding domains, and subsequent cell-cycle arrest, usually in G1 (24,26). Within this signal cascade the cyclin-dependent kinase (CDK) inhibitor p21WAF1 is an important mediator of the p53-induced cell-cycle arrest (26,27). Evidence that p53 is involved in DNA repair (28,29) and the induction of apoptosis (26,30) led to recognition that p53 participates in a signal transduction pathway which recognizes DNA damage and which can subsequently lead to growth arrest until DNA damage is repaired or programed cell death has been initiated (31). In contrast, cells which contain a mutated p53 gene, even if they express high constitutive levels of its protein product, lack a comparable response to DNA damage (21,24) and are more suceptible to the induction of mutations and development of transformed cell clones. The high prevalence of p53 gene mutations found in human cancers is consistent with this role for the p53 protein (32).
In order to determine how specific PAHDNA adduct levels determine the cellular response to PAHDNA damage, wild-type p53-expressing mammary adenocarcinoma-derived MCF-7 cells (33) were treated with (+)-syn- and ()-anti-DB[a,l]PDE and levels of DNA adducts in conjunction with those of the tumor suppressor protein p53 and the CDK inhibitor p21WAF1 were measured over a 96 h period after exposure.
 |
Materials and methods
|
---|
Chemicals
Nuclease P1 (EC 3.1.30.1; from Penicillium citrinum), human prostatic acid phosphatase (EC 3.1.3.2; from human semen), apyrase (EC 3.6.1.5; from Solanum tuberosum), phosphodiesterase I (EC 3.1.4.1; from Crotalus atrox) and proteinase K (EC 3.4.21.64; from Tritirachium album) were purchased from Sigma (St. Louis, MO). RNase T1 (EC 3.1.21.3; from Aspergillus oryzae) and RNase (DNase free, a heterogeneous mixture of ribonucleases from bovine pancreas) were obtained from Boehringer Mannheim (Indianapolis, IN). Unequilibrated phenol and cloned T4 polynucleotide kinase were purchased from United States Biochemical (Cleveland, OH). [
-33P]ATP [3500 Ci (129.5 TBq)/mmol] was purchased from Amersham (Arlington Heights, IL). The protease inhibitors leupeptin, phenylmethylsulfonyl fluoride (PMSF), aprotinin, and the trypsinchymotrypsin protease inhibitor (TCPI) were obtained from Boehringer Mannheim. Phosphate-buffered saline (PBS) contained 3.0 mM KCl, 1.5 mM KH2PO4, 140 mM NaCl, 8.0 mM Na2HPO4 (pH 7.4). Acrylamide and bisacrylamide for gel electrophoresis were purchased as a 40% mixture (w/v) from Bio-Rad (Hercules, CA). Preparation of enantiomeric 11,12-dihydrodiols of DB[a,l]P as described previously (17) allowed subsequent generation of optically pure (+)-syn- and ()-anti-DB[a,l]PDEs using the same synthetic route described for the racemic compounds (12).
Cell culture
The human mammary carcinoma cell line MCF-7 (original stock line was obtained from the Michigan Cancer Foundation) was grown in 175 cm2 cell culture flasks in a total volume of 50 ml of Dulbecco's modified Eagle's medium, high glucose type (DMEM with 4.5 g D-glucose/l; Gibco BRL, Grand Island, NY), supplemented with 10% fetal calf serum (FCS; Intergen, Purchase, NY), 0.1 mM non-essential amino acids (Gibco BRL) and 1 mM sodium pyruvate (Gibco BRL).
Treatment of MCF-7 cells with (+)-syn- and ()-anti-DB[a,l]PDE
After MCF-7 cells covered ~5060% of the surface area of the flasks (23 days after splitting of a confluent culture), the media was removed and the cells were washed twice with 20 ml sterile PBS. Medium without FCS (50 ml) was added to the flask, then 30 µl of a dimethyl sulfoxide (DMSO) solution of the enantiomerically pure (+)-syn- or ()-anti-DB[a,l]PDE was added. (Stock solutions of 1 mg/ml DB[a,l]PDE were diluted in DMSO to adjust the required concentration.) The cells were treated with the compounds in a concentration range between 0.001 and 0.1 µM. The control groups were treated with 30 µl DMSO alone. After 1 h of exposure, the medium was removed and replaced by medium containing 10% FCS. The cells were harvested at 2, 4, 6, 8, 12, 24, 48, 72 and 96 h after treatment by trypsinization with 0.05% trypsinEDTA (0.05% trypsin, 0.14 M NaCl, 3 mM KCl, 0.1 M Na2HPO4, 1.5 mM KH2PO4, 0.5 mM EDTA). After addition of an equal volume of medium containing 10% FCS, the cells were centrifuged at 1000 g, washed twice with PBS, and the cell pellet stored at 80°C.
DNA preparation
DNA isolation from MCF-7 cell pellets was carried out as described previously (10). Briefly, the cell pellets were homogenized in EDTAsodium dodecyl sulfate (SDS) buffer [10 mM Tris, 1 mM Na2EDTA, 1% SDS (w/v), pH 8] and incubated for 1 h at 37°C with RNase T1 (1000 U/ml) and RNase (DNase free; 5 µg/ml) on a shaker (100 r.p.m.). Then proteinase K (500 µg/ml) was added and the incubation continued for 1 h at 37°C. The mixture was extracted twice with 1 vol Tris-saturated phenol (1 M, pH 8.0) then twice with Tris-saturated phenol/chloroform/iso-amyl alcohol (25:24:1, v/v/v). The DNA was precipitated with 2 vol ethanol and 0.1 vol 5 M NaCl, washed with 70% ethanol, dried and dissolved in water. The DNA concentration in the solution was determined by A260 nm.
33P-post-labeling of DB[a,l]PDE-DNA adducts
Post-labeling was carried out as described previously (10). An aliquot of 10 µg DB[a,l]PDEDNA was digested, post-labeled with [
-33P]ATP (3500 Ci/mmol) and pre-purified with a Sep-Pak C18 cartridge (Waters, Milford, MA). Adducts were separated by HPLC on a C18 reverse-phase column (5 µm Ultrasphere ODS, 4.6x250 mm; Beckman Instruments) and the radiolabeled nucleotides measured with an on-line radioisotope flow-detector (Radiomatic FLO-ONE Beta; Packard Instruments, Downers Grove, IL). The level of DNA binding (reported as pmol adducts/mg DNA) was calculated based upon the efficiency of labeling of a B[a]P-7,8-dihydrodiol 9,10-epoxideDNA standard as described previously (34).
Isolation and western blotting of MCF-7 cell protein preparations
Total proteins from MCF-7 cells treated with (+)-syn- or ()-anti-DB[a,l]PDE were isolated according to the protocol described by Harlow and Lane (35). Briefly, the frozen cell pellet was diluted in an appropriate volume of RIPA lysis buffer [150 mM NaCl, 10 mM TrisHCl (pH 7.2), 1% sodium desoxycholate (w/v), 1% Triton X-100 (v/v), 0.1% SDS (w/v)] (~1 ml/107 cells). Prior to addition to the cells the RIPA buffer was pre-chilled to 4°C and the following were added per ml solution: 50 µl of 0.1 M Na2EDTA (pH 8.0) and 10 µl of each of the protease inhibitors leupeptin (1 mg/ml stock in water), PMSF (100 mM stock in iso-propanol), aprotinin (1 mg/ml stock in PBS) and TCPI (1 mg/ml stock in PBS). The buffercell mixture was aspirated through a fine-gauge needle (25 gauge fixed on a 1 ml syringe), then boiled in a water bath until an aggregate of sheared DNA and cell fragments had formed (~10 min). After cooling on ice, soluble proteins were separated by centrifugation at 10 000 g for 10 min. The protein concentration in this solution was spectrophotometrically determined at 562 nm using the Bicinchoninic acid colorimetric assay of Pierce (Rockford, IL). Lysates of A431 cells (human squamous carcinoma cell line) obtained from the Purdue University cell culture laboratory were also prepared for use as a p53-positive control in western blot analysis (36). These cells contain high amounts of p53 protein due to mutations in codons 248 and 273 of the corresponding gene which result in increased stability of the protein (37).
Prior to the western blotting, an appropriate amount of each isolated protein sample (40 µg) was diluted in loading buffer [10% glycerol (v/v), 5% 2-mercaptoethanol (v/v), 0.16 M Tris (pH 6.8), 3% SDS (w/v), 0.06% bromophenol blue (w/v)] and separated by SDSpolyacrylamide gel electrophoresis (PAGE) (0.1% SDS, 10% acrylamide) using the following electrode buffer: 1.44% glycine (w/v), 0.3% Tris base (w/v), 0.1% SDS (w/v), pH 8.3. After SDSPAGE, the proteins were transferred onto a nitrocellulose membrane (Bio-Rad) using a transfer buffer consisting of 1.44% glycine (w/v), 0.3% Tris base (w/v), 20% methanol (pH 8.3), then blocked at room temperature with Tris-buffered saline (TBS)Tween [150 mM NaCl, 0.01 M Tris (pH 8), 0.05% Tween-20 (v/v)] supplemented with 5% (w/v) non-fat dry milk powder for 10 min. After the blocking step had been repeated, the blot was incubated at room temperature for 1 h with the primary antibody diluted in TBSTween with 0.5% (w/v) non-fat dry milk powder. For p53 detection, the membrane was incubated with monoclonal antibody p53 Ab-2 (clone Ab 1801; Oncogene Science, Uniondale, NY) which recognizes both the human wild-type and mutant protein. The concentration used was 0.75 µg antibody/ml solution. Monoclonal antibody WAF 1 Ab-1 (clone EA10; Oncogene Science; 0.25 µg antibody/ml solution) was used for measuring the p21WAF1 protein. After incubation with the primary antibody the blot was washed twice with TBSTween for 10 min each and then incubated for 1 h at room temperature with the secondary antibody (goat anti-mouse IgG linked to horseradish peroxidase) diluted in TBSTween. The membranes were washed three times with TBSTween and the proteins were detected using the enhanced chemiluminescence technique (Amersham). Protein lysates from A431 cells were used as a positive control for p53.
 |
Results
|
---|
Human mammary carcinoma MCF-7 cells were treated with enantomerically pure (+)-syn- or ()-anti-DB[a,l]PDE (Figure 1
) in order to determine how DNA adduct formation affected the cellular content of the tumor suppressor p53 protein and the CDK-inhibitor p21WAF1. The HPLC elution profiles of the 33P-post-labeled DNA adducts formed in MCF-7 cells after treatment with 0.03 µM (+)-syn- and ()-anti-DB[a,l]PDE are shown in Figure 2
. The DNA from MCF-7 cells treated with (+)-syn-DB[a,l]PDE contained two major adduct peaks that eluted at 70 and 102 min. These have previously been identified as dA adducts by cochromatography with synthetic standards (10). The DNA from MCF-7 cells treated with ()-anti-DB[a,l]PDE contained three major adduct peaks. The large peak eluted at 78 min is a dA adduct and the smaller peaks eluted at 62 and 68 min are dG adducts (10). At all doses of these dihydrodiol epoxides tested, the proportions of the DNA adducts present were similar; however, the absolute amounts of DNA adducts varied with the dose of dihydrodiol epoxides.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 2. HPLC elution profiles of 33P-labeled DB[a,l]PDEDNA adducts obtained from DNA of MCF-7 cells exposed to 0.03 µM (+)-syn- or ()-anti-DB[a,l]PDE. Treatment with (+)-syn-DB[a,l]PDE resulted in two major DNA adducts that eluted at 70 and 102 min (dA adducts), whereas ()-anti-DB[a,l]PDE formed one predominant DNA adduct that eluted at 78 min (dA adduct) and two minor adducts at 62 and 68 min (dG adducts). All adducts were identified by cochromatography with synthetic standards as previously described (10). 33P-post-labeling and separation on HPLC was performed as described in Materials and methods.
|
|
The level of DNA binding and amount of p53 protein in MCF-7 cells observed 8 h after treatment with increasing concentrations of (+)-syn- and ()-anti-DB[a,l]PDE are shown in Figure 3
. For both dihydrodiol epoxides the amount of DNA adducts present increased with dose. Exposure to ()-anti-DB[a,l]PDE resulted in 3- to 4-fold higher DNA adduct formation than treatment with the same dose of (+)-syn-DB[a,l]PDE. An increase in p53 protein levels was also detected at low concentrations of ()-anti-DB[a,l]PDE. Whereas treatment with 0.01 µM ()-anti-DB[a,l]PDE caused a detectable increase in p53, the threshold dose for a visible increase in p53 was in a range between 0.02 and 0.03 µM in cells treated with the diastereomeric (+)-syn-DB[a,l]PDE (Figure 3
). The adduct level at which an increase in p53 levels was observed was in a comparable range of ~1520 pmol/mg DNA for both dihydrodiol epoxides.

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 3. DNA adduct and p53 protein levels in MCF-7 cells 8 h after exposure to (+)-syn- or ()-anti-DB[a,l]PDE. Detection of p53 by western blotting and analysis of total DNA binding by post-labeling was performed as described in Materials and methods. A431 (human squamous cell line) protein was included on the blot as a positive control for p53 protein (36).
|
|
Measurement of DNA adduct levels at different times after exposure to 0.01 and 0.05 µM (+)-syn- or ()-anti-DB[a,l]PDE also demonstrated the considerably greater amount of reaction of the ()-anti-diastereomer with DNA in these cells (Figure 4
). The maximal DNA adduct levels after treatment with 0.05 µM (+)-syn-DB[a,l]PDE or 0.01 µM ()-anti-DB[a,l]PDE were comparable (38 versus 35 pmol adducts/mg DNA). Almost 60 pmol adducts/mg DNA were obtained 6 h after incubation of MCF-7 cells with 0.05 µM ()-anti-DB[a,l]PDE, a modification level of ~1 adduct/50 000 nucleotides. DNA adduction reached maximal levels 68 h after exposure for both of the DB[a,l]PDE diastereomers used (Figure 4
). The amount of adducts subsequently decreased and reached 0.8 and 3.4 pmol/mg DNA 96 h after exposure to 0.01 µM (+)-syn- or ()-anti-DB[a,l]PDE (Figure 4
).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 4. Total DB[a,l]PDE-DNA binding in MCF-7 cells after exposure to (A) 0.01 µM and (B) 0.05 µM (+)-syn- or ()-anti-DB[a,l]PDE for the times indicated. Analysis of total DNA binding by post-labeling was performed as described in Materials and methods. Values represent the means of two independent experiments. Individual values varied from the mean within a range of ±35%.
|
|
Western blot analysis of p53 and p21WAF1 protein levels in MCF-7 cells after incubation with 0.01 µM (+)-syn- or ()-anti-DB[a,l]PDE are shown in Figure 5
. Levels of p53 exceeded control values between 4 and 6 h and reached the maximum level by 8 h after treatment with 0.01 µM ()-anti-DB[a,l]PDE. Subsequently, the amount decreased and returned to control values after 48 h. A large increase in p21WAF1 was observed ~8 h after treatment with ()-anti-DB[a,l]PDE (Figure 5
). The level of this protein remained elevated at all times examined up to 96 h. In contrast, no visible accumulation of the p53 protein was detected at any time after treatment of MCF-7 cells with 0.01 µM (+)-syn-DB[a,l]PDE (Figure 5
). Although treatment with 0.01 µM (+)-syn-DB[a,l]PDE did not cause a detectable increase in p53 protein, this treatment did result in a considerable increase in the amount of the p21WAF1 protein (Figure 5
). This increase was detectable after 12 h of exposure and persisted through 96 h.

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 5. p53 and p21WAF1 protein levels in MCF-7 cells exposed to 0.01 µM (+)-syn- or ()-anti-DB[a,l]PDE. At the times indicated treatment, harvesting, protein isolation and detection of p53 and p21WAF1 were performed as described in Material and methods. A431 (human squamous cell line) protein was included on the blot as a positive control for p53 protein (36). S, solvent (DMSO)-treated control cells.
|
|
To determine whether an intermediate dose of (+)-syn-DB[a,l]PDE that gave DNA binding levels comparable with that of 0.01 µM ()-anti-DB[a,l]PDE (Figure 3
) would result in similar effects on p53 and p21WAF1 levels, cells were treated with 0.025 µM (+)-syn-DB[a,l]PDE for up to 96 h. The western blots shown in Figure 6
demonstrate that this dose of (+)-syn-DB[a,l]PDE caused an increase in p53 at 68 h and a detectable increase in p21WAF1 at 12 h that persisted throughout 96 h. Thus, doses of (+)-syn- and ()-anti-DB[a,l]PDE that gave at least 15 pmol adducts/mg DNA caused similar increases in p53 and p21WAF1 protein levels.

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 6. p53 and p21WAF1 protein levels in MCF-7 cells exposed to 0.025 µM (+)-syn-DB[a,l]PDE. At the times indicated treatment, harvesting, protein isolation and detection of p53 and p21WAF1 were performed as described in Material and methods. A431 (human squamous cell line) protein was included on the blot as a positive control for p53 protein (36). S, solvent (DMSO)-treated control cells.
|
|
 |
Discussion
|
---|
The strong carcinogen DB[a,l]P has been found to exert its genotoxic activity in human mammary MCF-7 cells predominantly via metabolic activation to (+)-syn- and ()-anti-DB[a,l]PDE which react with genomic DNA to form mainly deoxyadenosine adducts (10,16). Measurement of DNA adducts formed in MCF-7 cells after direct incubation with each diastereomeric DB[a,l]PDE revealed that ()-anti-DB[a,l]PDE caused a 3- to 4-fold higher DNA modification level compared with (+)-syn-DB[a,l]PDE over a dose range from 0.005 to 0.1 µM (Figure 3
). Higher levels of ()-anti-DB[a,l]PDEDNA adducts compared with (+)-syn-DB[a,l]PDEDNA adducts were observed in cultures treated with 0.01 or 0.05 µM ()-anti- or (+)-syn-DB[a,l]PDE over the period of 296 h after exposure (Figure 4
). The significantly lower DNA adduct level observed after exposure to equimolar concentrations of (+)-syn-DB[a,l]PDE compared with ()-anti-DB[a,l]PDE might be due to increased sequestration of the syn-diastereomer as a consequence of its preferentially adopted alignedleft conformation (12). Vicinal syn-dihydrodiol epoxides preferring this conformation have been shown to undergo significantly accelerated solvolytic opening of their oxiranyl ring under neutral conditions compared with corresponding anti-diastereomers (38). This explanation would also be consistent with the observation that (+)-syn-DB[a,l]PDEDNA adducts were only detected in MCF-7 cells after treatment with high doses of the parent PAH (18 µM), whereas ()-anti-DB[a,l]PDEDNA adducts were present at detectable levels after exposure to a dose as low as 0.005 µM DB[a,l]P (39).
DNA damage induced by both DB[a,l]PDEs increased the cellular content of the p53 protein 8 h after exposure (Figure 3
). Therefore, DB[a,l]PDEDNA adducts caused a similar increase in p53 protein levels in MCF-7 cells as has been observed in various human cell cultures treated with a number of DNA-damaging agents including ionizing radiation (24), antitumor drugs (25) and metabolites of other PAHs (19,21,22). A dose as low as 0.01 µM ()-anti-DB[a,l]PDE caused a detectable increase in the level of the p53 protein, but 0.020.03 µM (+)-syn-DB[a,l]PDE was required to cause a comparable p53 increase (Figure 3
). Based upon the respective level of DNA adducts formed by these dihydrodiol epoxides (Figure 3
), (+)-syn-DB[a,l]PDE-DNA adducts were essentially as effective per adduct in causing an increase in p53 as ()-anti-DB[a,l]PDE-DNA adducts. Compared with the results obtained in a previous study using the ultimate genotoxic metabolite of B[a]P, B[a]P-7R,8S-dihydrodiol 9S,10R-epoxide {(+)-anti-B[a]PDE} (19), both fjord region DB[a,l]PDEs caused significantly higher DNA adduct and p53 protein levels/µM dihydrodiol epoxide. Exposure of MCF-7 cells to 0.03 µM ()-anti-DB[a,l]PDE (Figure 3
) or 0.3 µM (+)-anti-B[a]PDE (19), both stereoisomers with R,S,S,R-configuration, resulted in a comparable DNA binding (~50 pmol adducts/mg DNA) and increases in p53 protein levels. These findings indicate that irrespective of the structure of the specific PAHDNA adduct formed, doses of (+)-anti-B[a]PDE, ()-anti-DB[a,l]PDE or (+)-syn-DB[a,l]PDE that formed the same levels of adducts resulted in similar increases in p53 protein levels.
The effect of the DB[a,l]PDEs on cellular levels of p53 protein and p21WAF1 protein were similar at various times for doses that gave similar DNA adduct levels. Levels of p53 in MCF-7 cells increased to a detectable extent by 46 h after exposure to 0.01 µM ()-anti-DB[a,l]PDE (Figure 5
) or 0.025 µM (+)-syn-DB[a,l]PDE (Figure 6
). In both cultures a large increase in p21WAF1 protein was observed after 810 h and p21WAF1 levels remained elevated up to 96 h (Figures 5 and 6
). The time lag observed between p53 response and induction of p21WAF1 after treatment with 0.01 µM ()-anti-DB[a,l]PDE or 0.025 µM (+)-syn-DB[a,l]PDE is consistent with a temporal connection between these increases. Others have demonstrated in various wild-type p53-expressing human cell lines that DNA damage leads to nuclear accumulation of this protein followed by induction of p21WAF1 and subsequent cell-cycle arrest in G1 (24,26,27,40). In addition to this p53-dependent signal transduction pathway via induction of p21WAF1, evidence has been found for p53-independent induction of p21WAF1 caused by DNA damage (41). The results obtained after treatment of MCF-7 cells with 0.01 µM (+)-syn-DB[a,l]PDE (Figure 5
) may involve such a pathway. Although no increase in p53 was observed at any time after treatment up to 96 h, a considerable increase in p21WAF1 protein was detected by 12 h after exposure and maintained for 4 days.
The CDK-inhibitor p21WAF1 inhibits both the cyclin-dependent G1 kinases and the G2/M-specific cdc2 kinase (42,43). A number of types of DNA damage have been demonstrated to cause G1 arrest controlled by a wild-type p53-dependent induction of p21WAF1 (21,24,44). However, DNA damage can also result in an arrest in G2. DNA damage induced by
-irradiation has been found to induce G2/M accumulation of cells that lack wild-type p53 expression (24,45). Up-regulation of wild-type p53 gene expression in human fibroblasts in the absence of any DNA damaging agent has also been shown to result in the mediation of a reversible growth arrest by both the control of the G1 and the G2/M checkpoints (45). In both cases, the arrest was associated with high levels of p21WAF1 (45).
Measurement of the p21WAF1 content in MCF-7 cells after treatment with 0.005 µM DB[a,l]P (39) or different doses of ()-anti- and (+)-syn-DB[a,l]PDE (Figures 5 and 6
) revealed that the level of this protein did not exceed control values until ~48 h after exposure to the parent PAH or 1224 h after exposure to both fjord region DB[a,l]PDEs. Therefore, any cell-cycle arrest caused by increases in p21WAF1 in response to the DB[a,l]PDE-induced DNA damage may occur after replication of DNA containing appreciable levels of DB[a,l]PDEDNA adducts. The concept that PAHs can act as `stealth carcinogens' by allowing replication prior to cell-cycle arrest has been proposed by Khan et al. (22). Although they observed cell-cycle arrest in S phase with only a small increase in p21WAF1 protein levels in cells treated with racemic anti-11,12-dihydrodiol 13,14-epoxide of benzo[g]chrysene, the 21 h time-point tested may have been early in the p21WAF1 response (22). In studies with DB[a,l]P (39), B[a]P and (+)-anti-B[a]PDE (19; L.C.Kaspin and W.M.Baird, unpublished results) we observed a cell-cycle arrest in G2/M in MCF-7 cells and a large increase in p21WAF1 levels. The observed cell-cycle arrest in phases other than G1 may be due to a DNA damage-induced long-term expression of p21WAF1, such as described by Di Leonardo et al. (44), caused by treatment with DB[a,l]P or (+)-syn- and ()-anti-DB[a,l]PDE (Figures 5 and 6
).
The present study demonstrates that there is a dosedependent increase in p53 protein levels in MCF-7 cells after exposure to (+)-syn- or ()-anti-DB[a,l]PDE. Both stereoisomeric compounds are the DNA-binding products of metabolic activation of DB[a,l]P. The stereoisomer with R,S,S,R-configuration, the ()-anti-DB[a,l]PDE, forms significantly more DNA adducts and induces an increase of p53 at significantly lower concentrations than the (+)-syn-DB[a,l]PDE with S,R,S,R-configuration. These results together with our previous findings on (+)-anti-B[a]PDE-treated MCF-7 cells (19) indicate that the increase of wild-type p53 protein levels is related to formation of a critical level of adducts rather than by specific adduct structures and configurations. The results demonstrate that formation of DB[a,l]PDEDNA adducts also causes a long-term induction of the CDK inhibitor p21WAF1. The presence of an increase in p21WAF1 protein levels in the absence of a detectable increase in p53 protein levels in cells treated with 0.01 µM (+)-syn-DB[a,l]PDE suggests that p53-independent induction of p21WAF1 can also result from DB[a,l]PDEDNA adduct formation. The long term induction of p21WAF1 after formation of DB[a,l]PDEDNA adducts may lead to G2/M arrest, which remains to be further established.
 |
Acknowledgments
|
---|
This work was financially supported by grants CA40228 and CA28825 from the National Cancer Institute, Department of Health and Human Services (W.M.B.), and by the Deutsche Forschungsgemeinschaft (SFB 302) (A.S.).
 |
Notes
|
---|
6 To whom correspondene should be addressed Email: william.baird{at}orst.edu 
 |
References
|
---|
-
Cavalieri,E.L., Higginbotham,S., RamaKrishna,N.V.S., Devanesan,P.D., Todorovic,R., Rogan,E.G. and Salmasi,S. (1991) Comparative doseresponse tumorigenicity studies of dibenzo[a,l]pyrene versus 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene and two dibenzo[a,l]pyrene dihydrodiols in mouse skin and rat mammary gland. Carcinogenesis, 12, 19391944.[Abstract]
-
Higginbotham,S., RamaKrishna,N.V.S., Johansson,S.L., Rogan,E.G. and Cavalieri,E.L. (1993) Tumor-initiating activity and carcinogenicity of dibenzo[a,l]pyrene versus 7,12-dimethylbenz[a]anthracene and benzo-[a]pyrene at low doses in mouse skin. Carcinogenesis, 14, 875878.[Abstract]
-
LaVoie,E.J., He,Z.-M., Meegalla,R.L. and Weyand,E.H. (1993) Exceptional tumor-initiating activity of 4-fluorobenzo[j]fluoranthene on mouse skin: comparison with benzo[j]fluoranthene, 10-fluoro-benzo[j]fluoranthene, benzo[a]pyrene, dibenzo[a,l]pyrene and 7,12-dimethylbenz[a]anthracene. Cancer Lett., 70, 714.[ISI][Medline]
-
DeRatt,W.K., Kooijman,S.A.L.M. and Gielen,J.W.J. (1987) Concentrations of polycyclic hydrocarbons in airborne particles in the Netherlands and their correlation with mutagenicity. Sci. Total. Environ., 66, 95114.[ISI][Medline]
-
Kozin,I.S., Gooijer,C. and Velthorst,N.H. (1995) Direct determination of dibenzo[a,l]pyrene in crude extracts of environmental samples by laser excited Shpol'skii spectroscopy. Anal. Chem., 67, 16231626.[ISI]
-
Mumford,J.L., Harris,D.B., Williams,K., Chuang,J.C. and Cooke,M. (1987) Indoor air sampling and mutagenicity studies of emissions from unvented coal combustion. Environ. Sci. Technol., 21, 308311.[ISI]
-
Mumford,J.L., Xueming,L., Fuding,H., Xu,B.L. and Chuang,J.C. (1995) Human exposure and dosimetry of polycyclic aromatic hydrocarbons in urine from Xuan Wei, China with high lung cancer mortality associated with exposure to unvented coal smoke. Carcinogenesis, 16, 30313036.[Abstract]
-
Snook,M.E., Severson,R.F., Arrendale,R.F., Higman,H.C. and Chortyk,O.T. (1977) The identification of high molecular weight polynuclear aromatic hydrocarbons in a biologically active fraction of cigarette smoke condensate. Beitr. Tabakforsch., 9, 79101.[ISI]
-
Ralston,S.L., Lau,H.H.S., Seidel,A., Luch,A., Platt,K.L. and Baird,W.M. (1994) Identification of dibenzo[a,l]pyreneDNA adducts formed in cells in culture and in mouse skin. Polycyclic Aromat. Comp., 6, 199206.
-
Ralston,S.L., Seidel,A., Luch,A., Platt,K.L. and Baird,W.M. (1995) Stereoselective activation of dibenzo[a,l]pyrene to ()-anti(11R, 12S,13S,14R)- and (+)-syn(11S,12R,13S,14R)-11,12-diol-13,14-epoxides which bind extensively to deoxyadenosine residues of DNA in the human mammary carcinoma cell line MCF-7. Carcinogenesis, 16, 28992907.[Abstract]
-
Li,K.-M., Todorovic,R., Rogan,E.G., Cavalieri,E.L., Ariese,F., Suh,M., Jankowiak,R. and Small,G.J. (1995) Identification and quantitation of dibenzo[a,l]pyreneDNA adducts formed by rat liver microsomes in vitro: preponderance of depurination adducts. Biochemistry, 34, 80438049.[ISI][Medline]
-
Luch,A., Glatt,H.-R., Platt,K.L., Oesch,F. and Seidel,A. (1994) Synthesis and mutagenicity of the diastereomeric fjord-region 11,12-dihydrodiol 13,14-epoxides of dibenzo[a,l]pyrene. Carcinogenesis, 15, 25072516.[Abstract]
-
Gill,H.S., Kole,P.L., Wiley,J.C., Li,K.M., Higginbotham,S., Rogan,E.G. and Cavalieri,E.L. (1994) Synthesis and tumor-initiating activity in mouse skin of dibenzo[a,l]pyrene syn- and anti-fjord-region diolepoxides. Carcinogenesis, 15, 24552460.[Abstract]
-
Amin,S., Krzeminski,J., Rivenson,A., Kurtzke,C., Hecht,S.S. and El-Bayoumy,K. (1995) Mammary carcinogenicity in female CD rats of fjord region diol epoxides of benzo[c]phenanthrene, benzo[g]chrysene and dibenzo[a,l]pyrene. Carcinogenesis, 16, 19711974.[Abstract]
-
Amin,S., Desai,D., Dai,W., Harvey,R.G. and Hecht,S.S. (1995) Tumorigenicity in newborn mice of fjord region and other sterically hindered diol epoxides of benzo[g]chrysene, dibenzo[a,l]pyrene (dibenzo[def,p]chrysene), 4H-cyclopenta[def]chrysene and fluoranthene. Carcinogenesis, 16, 28132817.[Abstract]
-
Melendez-Colon,V.J., Smith,C.A., Seidel,A., Luch,A., Platt,K.-L. and Baird,W.M. (1997) Formation of stable adducts and absence of depurination DNA adducts in cells and DNA treated with the potent carcinogen dibenzo[a,l]pyrene or its diol epoxides. Proc. Natl Acad. Sci. USA, 94, 1354213547.[Abstract/Free Full Text]
-
Luch,A., Seidel,A., Glatt,H.-R. and Platt,K.L. (1997) Metabolic activation of the (+)-S,S- and ()-R,R-enantiomers of trans-11,12-dihydroxy-11,12-dihydrodibenzo[a,l]pyrene: stereoselectivity, DNA adduct formation, and mutagenicity in Chinese hamster V79 cells. Chem. Res. Toxicol., 10, 11611170.[ISI][Medline]
-
Ralston,S.L., Coffing,S.L., Seidel,A., Luch,A., Platt,K.L. and Baird,W.M. (1997) Stereoselective activation of dibenzo[a,l]pyrene and its trans-11,12-dihydrodiol to fjord region 11,12-diol 13,14-epoxides in a human mammary carcinoma MCF-7 cell-mediated V79 cell mutation assay. Chem. Res. Toxicol., 10, 687693.[ISI][Medline]
-
Kaspin,L.C. and Baird,W.M. (1996) Anti-benzo[a]pyrene-7,8-diol-9,10-epoxide treatment increases levels of the proteins p53 and p21WAF1 in the human mammary carcinoma cell line MCF-7. Polycyclic Aromat. Comp., 10, 299306.[ISI]
-
Bjelogrlic,N., Mäkinen,M., Stenbäck,F. and Vähäkangas,K. (1994) Benzo[a]pyrene-7,8-diol-9,10-epoxideDNA adducts and increased p53 protein in mouse skin. Carcinogenesis, 15, 771774.[Abstract]
-
Rämet,M., Castrén,K., Järvinen,K., Pekkala,K., Turpeenniemi-Hujanen,T., Soini,Y., Pääkkö,P. and Vähäkangas,K. (1995) p53 protein expression is correlated with benzo[a]pyreneDNA adducts in carcinoma cell lines. Carcinogenesis, 16, 21172124.[Abstract]
-
Khan,Q.A., Vousden,K.H. and Dipple,A. (1997) Cellular response to DNA damage from a potent carcinogen involves stabilization of p53 without induction of p21waf1/cip1. Carcinogenesis, 18, 23132318.[Abstract]
-
Yuan,J., Yeasky,T.M., Havre,P.A. and Glazer,P.M. (1995) Induction of p53 in mouse cells decreases mutagenesis by UV radiation. Carcinogenesis, 16, 22952300.[Abstract]
-
Kastan,M.B., Onyekwere,O., Sidransky,D., Vogelstein,B. and Craig,R.W. (1991) Participation of p53 protein in the cellular response to DNA damage. Cancer Res., 51, 63046311.[Abstract]
-
Fritsche,M., Haessler,C. and Brandner,G. (1993) Induction of nuclear accumulation of the tumor-suppressor protein p53 by DNA-damaging agents. Oncogene, 8, 307318.[ISI][Medline]
-
El-Deiry,W.S., Harper,J.W., O'Connor,P.M. et al. (1994) WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res., 54, 11691174.[Abstract]
-
Harper,J.W., Adami,G.R., Wei,N., Keyomarsi,K. and Elledge,S.J. (1993) The p21 cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell, 75, 805816.[ISI][Medline]
-
Marx,J. (1994) New link found between p53 and DNA repair. Science, 266, 13211322.[ISI][Medline]
-
Ford,J.M. and Hanawalt,P.C. (1995) Li-Fraumeni syndrome fibroblasts homozygous for p53 mutations are deficient in global DNA repair but exhibit normal transcription-coupled repair and enhanced UV resistance. Proc. Natl Acad. Sci. USA, 92, 88768880.[Abstract]
-
Shaw,P., Bovey,R., Tardy,S., Sahli,R., Sordat,B. and Costa,J. (1992) Induction of apoptosis by wild-type p53 in a human colon tumor-derived cell line. Proc. Natl Acad. Sci. USA, 89, 44954499.[Abstract]
-
Hartwell,L.H. and Kastan,M.B. (1995) Cell cycle control and cancer. Science, 266, 18211828.[ISI]
-
Harris,C.C. and Hollstein,M. (1993) Clinical implications of the p53 tumor-suppressor gene. N. Engl. J. Med., 329, 13181327.[Free Full Text]
-
Runnebaum,I.B., Nagarajan,M., Bowman,M., Soto,D. and Sukumar,S. (1991) Mutations in p53 as potential molecular markers for human breast cancer. Proc. Natl Acad. Sci. USA, 88, 1065710661.[Abstract]
-
Lau,H.H.S. and Baird,W.M. (1991) Detection and identification of benzo[a]pyreneDNA adducts by [35S]phosphorothioate labeling and HPLC. Carcinogenesis, 12, 885893.[Abstract]
-
Harlow,E. and Lane,D. (1988) AntibodiesA Laboratory Manual. Sigma, Cold Spring Harbor Laboratory, NY.
-
Barnes,D.W. (1984) Growth characteristics of A431 human epidermoid carcinoma cells in serum-free medium: inhibition by epidermal growth factor. Adv. Exp. Med. Biol., 172, 4966.[ISI][Medline]
-
Reiss,M., Brash,D.E., Munoz-Antonia,T., Simon,J.A., Ziegler,A., Vellucci,V.F. and Zhou,Z.-L. (1992) Status of the p53 tumor suppressor gene in human squamous carcinoma cell lines. Oncology Res., 4, 349357.[ISI][Medline]
-
Sayer,J.M., Yagi,H., Silverton,J.V., Friedman,S.L., Whalen,D.L. and Jerina,D.M. (1982) Conformational effects in the hydrolyses of rigid benzylic epoxides: implications for diol epoxides of polycyclic hydrocarbons. J. Am. Chem. Soc., 104, 19721978.[ISI]
-
Baird,W.M., Kaspin,L.C., Kudla,K., Seidel,A., Greim,H. and Luch,A. (1999) Relationship of dibenzo[a,l]pyreneDNA binding to the induction of p53, p21WAF1 and cell-cycle arrest in human cells in culture. Polycyclic Aromat. Comp., in press.
-
El-Deiry,W.S., Tokino,T., Velculescu,V.E., Levy,D.B., Parsons,R., Trent,J., Lin,D., Mercer,W.E., Kinzler,K.W. and Vogelstein,B. (1993) WAF1, a potential mediator of p53 tumor suppression. Cell, 75, 817825.[ISI][Medline]
-
Sheikh,M.S., Li,X.-S., Chen,J.-C., Shao,Z.-M., Ordonez,J.V. and Fontana,J.A. (1994) Mechanisms of regulation of WAF1/Cip1 gene expression in human breast carcinoma: role of p53-dependent and independent signal transduction pathways. Oncogene, 9, 34073415.[ISI][Medline]
-
Nurse,P. (1990) Universal control mechanism regulating onset of M-phase. Nature, 344, 503508.[ISI][Medline]
-
Xiong,Y., Hannon,G.J., Zhang,H., Casso,D., Kobayashi,R. and Beach,D. (1993) p21 is a universal inhibitor of cyclin kinases. Nature, 366, 701704.[ISI][Medline]
-
Di Leonardo,A., Linke,S.P., Clarkin,K. and Wahl,G.M. (1994) DNA damage triggers a prolonged p53-dependent G1 arrest and long-term induction of Cip1 in normal human fibroblasts. Genes Dev., 8, 25402551.[Abstract]
-
Agarwal,M.L., Agarwal,A., Taylor,W.R. and Stark,G.R. (1995) p53 controls both the G2/M and the G1 cell cycle checkpoints and mediates reversible growth arrest in human fibroblasts. Proc. Natl Acad. Sci. USA, 92, 84938497.[Abstract]
Received September 15, 1998;
revised December 23, 1998;
accepted January 26, 1999.