Effects of the (–)-anti-11R,12S-dihydrodiol 13S,14R-epoxide of dibenzo[a,l]pyrene on DNA adduct formation and cell cycle arrest in human diploid fibroblasts

Brinda Mahadevan1, Andreas Luch1,2, Albrecht Seidel3, Jill C. Pelling4 and William M. Baird1,5

1 Department of Environmental and Molecular Toxicology, Agricultural and Life Sciences 1011, Oregon State University, Corvallis, OR 97331-7302, USA,
2 Institute of Toxicology and Environmental Hygiene, Technical University of Munich, 80636 Munich, Germany,
3 Biochemical Institute for Environmental Carcinogens, Lurup 4, 22927 Grosshansdorf, Germany and
4 Department of Pathology and Laboratory Medicine, The University of Kansas Medical Center, Kansas City, KS, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The tumor suppressor protein p53 plays an important role in recognition of DNA damage and induction of subsequent cell cycle arrest. One of its target genes encodes the protein p21WAF1, which is involved in mediation of growth arrest after DNA damage has occurred. Dibenzo[a,l]pyrene (DB[a,l]P) is a polycyclic aromatic hydrocarbon which is an exceptionally potent carcinogen. A reactive secondary metabolite of DB[a,l]P, the fjord region (–)-anti-11R,12S-dihydrodiol 13R,14S-epoxide [(–)-anti-DB[a,l]PDE] was used to investigate DNA damage via adduct formation and cell cycle arrest in human diploid fibroblast cell cultures (HDF). Synchronous HDF were exposed to increasing concentrations (0.014, 0.028 and 0.07 µM) of (-)-anti-DB[a,l]PDE and at 1, 12, 24 and 42 h after treatment cell pellets were analyzed for DNA adduct formation and cell cycle arrest. Exposure of HDF to 0.07 µM (–)-anti-DB[a,l]PDE caused a total DNA binding level of 113 pmol adducts/mg DNA (42 h after treatment). G1 arrest was induced by this treatment, with 91% of the cells remaining in G1 phase compared with the solvent-treated control cultures (50%) as analyzed by propidium iodide staining and flow cytometry. Further investigation of the percentage of cells in S phase by 5-bromo-2'-deoxyuridine incorporation confirmed the G1 arrest in HDF treated with 0.07 µM (–)-anti-DB[a,l]PDE, with only 1.5% of the cells moving into S phase compared with 39% in the control 42 h after treatment. Induction of p53 and p21WAF1 was demonstrated by western blot analysis.

Abbreviations: B[a]P, benzo[a]pyrene; BPDE, 7,8-dihydrodiol 9,10-epoxide of B[a]P; BrdU, 5-bromo-2'-deoxyuridine; DB[a,l]P, dibenzo[a,l]pyrene; DB[a,l]PDE, 11,12-dihydrodiol 13,14-epoxide of DB[a,l]P; HDF, human diploid fibroblast cell cultures; NER, nucelotide excision repair; PAH, polycyclic aromatic hydrocarbon; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The tumor suppressor protein p53 is a nuclear phosphoprotein involved in at least three different physiological processes, cell cycle arrest, DNA repair and apoptosis (13), which contribute together in an interconnected manner to the function of p53 in suppressing the growth of cancer cells (4,5). Consequently, mutations within the p53 gene which render the corresponding protein inactive are the most common alterations detected in human tumor cells (6).

Cells containing wild-type p53 protein are able to recognize DNA damage induced by ionizing radiation (2), UV light (7) or other DNA damaging agents such as antitumor drugs (8) and polycyclic aromatic hydrocarbons (PAHs) (9,10). PAHs are products of incomplete combustion of organic matter and many of them are potent carcinogens widespread in the environment (11). The immediate cellular responses upon exposure to these DNA damaging agents consist of nuclear accumulation of p53, transcriptional induction of various target genes, such as the cyclin dependent kinase inhibitor p21WAF1, and subsequent cell cycle arrest in G1 (2,12,13,14). In contrast, cells that express mutated or high constitutive levels of p53 protein lack a comparable response to DNA damage (2,9). These observations suggested that p53 participates in a signal transduction pathway which recognizes altered genomic DNA and subsequently leads to growth arrest until DNA repair or programmed cell death (apoptosis) has occurred (1,3,15). Both events would prevent manifestation of genomic mutations and, therefore, the potential development of transformed cell clones and the generation of cancer. Exposure to benzo[a]pyrene (B[a]P) has been found to increase p53 protein levels in vivo (10,16) and in cell cultures (9,17). Induction of p53 and p21WAF1 and cell cycle arrest in G2 upon exposure to dibenzo[a,l]pyrene (DB[a,l]P) has been demonstrated in human MCF-7 cells (18). Subsequently, Luch et al. (19) demonstrated that the level of DNA modification by the ultimate mutagens, the 11,12-dihydrodiol 13,14-epoxides of DB[a,l]P, determined the effect on the proteins p53 and p21WAF1 in human MCF-7 cells.

DB[a,l]P is a considerably more potent carcinogen in rodent bioassays than B[a]P (2022). DB[a,l]P is derived from the bay region PAH B[a]P by condensation of an additional benzo ring at the 11,12-position. This creates a sterically hindered fjord region and the hexacyclic system of DB[a,l]P (Figure 1Go). Knowledge about the biological potency of DB[a,l]P, together with its detection in the environment (23,24), has prompted several investigations on the biotransformation of this compound (2527). Metabolism and DNA adduct formation studies using cytochrome P450-containing liver microsomes (25,26) or human MCF-7 cells (27) revealed that DB[a,l]P is converted to its (+)-syn- and (–)-anti-11,12-dihydrodiol 13,14-epoxides (DB[a,l]PDE), which predominantly mediate the genotoxicity of the parent compound via binding at deoxyadenosine residues in DNA (Figure 1Go; ref. 27).



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Fig. 1. Schematic representation of the stereoselective metabolism of and DNA adduct formation by DB[a,l]P) in human mammary carcinoma MCF-7 cells. The main DNA adducts found were generated by binding of 11,12-dihydrodiol 13,14-epoxides at dA residues in DNA (27).

 
In order to gain insight into the molecular response to DNA damage caused by covalently bound metabolites of DB[a,l]P (Figure 1Go) we examined hydrocarbon–DNA interactions and their effects on cell signaling through the p53 pathway and cell cycle checkpoints in synchronized human diploid fibroblast cell cultures (HDF). These HDF were selected for this study as they are readily synchronized in culture by density-dependant growth arrest without the use of metabolic blocking agents and, being an early passage human culture, they retain the signal transduction pathways that may have been altered in established cell lines.


    Materials and methods
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Chemicals
Nuclease P1 (from Penicillium citrinum, EC 3.1.30.1), human prostatic acid phosphatase (from human semen, EC 3.1.3.2), apyrase (from Solanum tuberosum, EC 3.6.1.5), phosphodiesterase I (from Crotalus atrox, EC 3.1.4.1) and proteinase K (from Tritirachium album, EC 3.4.21.64) were purchased from Sigma Chemical Co. (St Louis, MO). RNase T1 (from Asperigillus oryzae, EC 3.1.21.3) and DNase-free RNase (a heterogeneous mixture of ribonucleases from bovine pancreas) were obtained from Boehringer Mannheim Co. (Indianapolis, IN). Unequilibrated phenol and cloned T4 polynucleotide kinase were purchased from US Biochemical (Cleveland, OH). [{gamma}-33P]ATP (3500 Ci, 129.5 TBq/mmol) was purchased from Amersham Corp. (Arlington Heights, IL). The protease inhibitors leupeptin, phenylmethylsulfonyl fluoride (PMSF) and aprotinin were obtained from Boehringer Mannheim Corp. (Indianapolis, IN). 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 Laboratories (Hercules, CA). Preparation of enantiomeric 11,12-dihydrodiols of DB[a,l]P as described previously (28) allowed subsequent generation of optically pure (+)-syn- and (–)-anti-DB[a,l]PDEs using the same synthetic route described for the racemic compounds (29).

Cell synchronization and treatment
The HDF were held at confluency for 3 days to bring them to G0 phase. They were split in the ratio 1:5 and 18 h later treated in the re-entered G1 phase with increasing concentrations (0.014, 0.028 and 0.07 µM) of (–)-anti-DB[a,l]PDE for 1–2 h in serum-free medium, followed by replacement with medium containing 10% serum for various incubation periods. The cells were harvested at 1, 12, 24 and 42 h after treatment for cell cycle analysis and DNA adduct formation. Treatment was carried out separately for individual analyses and the experiments were repeated at least twice and many three or four times. A representative plot is shown in Results.

DNA isolation from HDF
DNA isolation from HDF was carried out as described previously (30). Briefly, the prepared cell pellet was resuspended in 1 ml of lysis buffer (10 mM Tris, pH 8.0, 0.1 mM EDTA, pH 8.0, 20 µg/ml RNase A, 0.5% SDS) and incubated at 37°C for 1 h. Proteinase K (100 µg/ml) was then added and incubated at 50°C for at least 4 h to overnight. The mixture was then extracted with an equal volume of phenol equilibrated with 0.5 M Tris, pH 8.0. The phases were mixed gently in rocking tubes for 5–10 min and separated by centrifugation at 5000 g for 15 min at room temperature. The aqueous phase was then transferred to a clean tube. Extractions were performed twice if the interface was not clear. The DNA was precipitated with 5 M NaCl (final concentration of 0.2 M) and 2 vol. 100% ethanol, washed with 70% ethanol, dried and dissolved in water. The DNA concentration in the solution was determined spectrophotometrically at 260 nm.

Protein isolation from HDF
Total proteins from asynchronous HDF treated with (–)-anti-DB[a,l]PDE were isolated according to the following protocol. Treated cells were harvested by trypsinization and centrifugation at specific times as described in Results. After harvest the cells were rinsed with PBS at least twice and PBS was suctioned off after the last rinse. The cells were then suspended in 300 µl of Triton lysis buffer (20 mM Tris, pH 7.4, 137 mM NaCl, 25 mM ß-glycerophosphate, 2 mM EDTA, 1 mM Na3VO4, 2 mM sodium pyrophosphate, 1% Triton X-100 and 10% glycerol) and then held on ice. Protease inhibitors (1 mM PMSF, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 2 mM benzamidine and 0.5 mM dithiothreitol) were added to the Triton lysis buffer just before use. The cell lysate collected was held on ice for at least 30 min with intermittant mixing, then centrifuged at 4°C at 14 000 g. The supernatant was stored at –20°C in aliquots. The protein concentration in the aliquots was spectrophotometrically determined at 562 nm using the bicinchoninic acid colorimetric assay (Pierce, Rockford, IL).

Immunoblotting
Prior to immunoblotting an appropriate amount of each isolated protein sample (40 µg) was diluted in loading buffer and separated by SDS–PAGE. After SDS–PAGE the proteins were transferred onto a nitrocellulose membrane (Bio-Rad). For p53 detection the membrane was incubated with monoclonal antibody p53 Ab-2 (clone Ab 1801; Oncogene Science, Uniondale, NY). The concentration used was 0.75 µg antibody/ml solution. Monoclonal antibody WAF1 Ab-1 (clone EA10; Oncogene Science) at a concentration of 0.25 µg antibody/ml solution was used to measure p21WAF1 protein. After incubation with the primary antibody the blot was washed twice with PBS/Tween for 5 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 PBS/Tween. The membranes were washed three times with PBS/Tween and the proteins detected using the enhanced chemiluminescence technique (Amersham). Lysates of A431 cells (human squamous carcinoma cell line) obtained from Oregon State University Cell Culture Laboratory were also prepared for use as a p53-positive control in western blot analyses (31,32). 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 (33).

33P-post-labeling of DB[a,l]PDE–DNA adducts
Post-labeling was carried out as described previously (19,34). An aliquot of 10 µg DB[a,l]PDE-treated DNA was digested, post-labeled with [{gamma}-33P]ATP and pre-purified with a Sep-Pak C18 cartridge (Waters, Milford, MA). Adducts were separated by HPLC 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 on labeling of a [3H]B[a]PDE–DNA standard of known modification level which was analyzed together with each set of post-labeling samples (34). Two independent sets of post-labeling reactions were carried out for every sample treated, to determine the total PAH–DNA adduct levels.

Cell cycle analysis
The method used was that published by Dolbeare et al. (35). Briefly, synchronous HDF were incubated with 20 µM 5-bromo-2'-deoxyuridine (BrdU), then harvested at a specified time after treatment and fixed with ice-cold ethanol (70%). Then cellular DNA was denatured by incubating the cells with 2 N HCl, 0.5% Triton X-100. Upon adjusting the pH to 7.0 with 2 M Tris, pH 8.0, and resuspension in PBS the cells were incubated for 30 min with a solution containing 4 µg anti-BrdU antibody/ml PBS (Oncogene Science), 5% fetal bovine serum and 0.1% sodium azide, washed with PBS and subsequently incubated for 30 min with fluorescein isothiocyanate-linked secondary antibodies (10 µg/ml PBS; Oncogene Science). After staining of cellular DNA with Vindelov's solution (10 mg/ml RNase A, 1% Triton X-100, 500 µg/ml propidium iodide in PBS) at 4°C for at least 30 min the DNA content was determined on an EPICS flow cytometer (Hialeah, FL). The percentages of cells in the G1, S and G2/M phases of the cell cycle were determined using the Winlist (Verity Software House, Topsham, ME) and Multicycle (Phoenix Flow Systems, San Diego, CA) computer programs.


    Results
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 Materials and methods
 Results
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Cell cycle analysis after exposure of synchronized HDF to (–)-anti-DB[a,l]PDE
In order to examine the effect of PAH–DNA adducts in inducing cell cycle arrest in synchronized HDF, we determined the proportion of cells in the G1, S and G2 phases, respectively, by fixing and staining the cells with propidium iodide after treatment and analysing the fractions by flow cytometry. The histograms obtained after multicycle analysis 42 h after treatment of HDF with increasing concentrations of (–)-anti-DB[a,l]PDE are shown in Figure 2Go. Upon treatment with increasing concentrations of (–)-anti-DB[a,l]PDE the percentage of cells that remained in G1 increased from 50 (control) to 91% [0.07 µM (–)-anti-DB[a,l]PDE] (Figure 2Go), indicating arrest in the G1 phase of the cell cycle. Apoptosis analysis by Annexin V staining and flow cytometry confirmed that cytotoxicity was not the cause of cell death and that viable cells were actually arresting in G1. There was no difference in the percentage of viable cells between the control and cells treated with a dose of 0.014 µM (-)-anti-DBPDE for 42 h.



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Fig. 2. Histograms showing the percentage of HDF in different phases of the cell cycle, 42 h after treatment with (-)-anti-DB[a,l]PDE. Cells were stained with propidium iodide and analyzed by flow cytometry.

 
Cell cycle analysis performed on cells collected from 1 to 42 h after treatment and the corresponding percentage of cells in each phase are detailed graphically in Figure 3Go. Treatment with increasing concentrations of (–)-anti-DB[a,l]PDE (0.014–0.07 µM) resulted in growth arrest in the G1 phase depending on time (Figures 2 and 3GoGo). When the cells were treated with a low dose of 0.014 µM (–)-anti-DB[a,l]PDE the percentage of cells in G1 followed the same trend as seen in the solvent-treated control (Figure 3Go). In contrast, exposure to 0.028 or 0.07 µM (–)-anti-DB[a,l]PDE resulted in >80% of the cells arrested in G1 phase 24 and 42 h after treatment (Figure 3Go).



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Fig. 3. Graphical representation of the percentage of HDF in different phases of the cell cycle, after treatment with increasing concentrations of DB[a,l]PDE over time. The cells were stained with propidium iodide and analyzed by flow cytometry. {blacklozenge}, Control; •, 0.014 µM; {blacktriangleup}, 0.028 µM; {blacksquare}, 0.07 µM.

 
Further examination of the percentage of cells in S phase by BrdU incorporation confirmed the G1 arrest of HDF. Histograms of the treated and control cells at different time points are graphically detailed in Figure 4Go. Forty-two hours after treatment with 0.028 and 0.07 µM (–)-anti-DB[a,l] PDE only 1.5% of the cells had moved into S phase, compared with 39% in the control. However, the lower dose of 0.014 µM (–)-anti-DB[a,l]PDE showed a similar trend to that seen in the control 42 h after treatment (Figure 4Go).



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Fig. 4. Graphical representation of the percentage of HDF in different phases of the cell cycle, after treatment with increasing concentrations of (-)-anti-DB[a,l]PDE over time. The cells were analyzed by flow cytometry, especifically for S phase cells showing incorporation of BrdU. {lozenge}, Control; {circ}, 0.014 µM; {triangleup}, 0.028 µM; {square}, 0.07 µM.

 
DNA adduct formation in synchronized HDF after exposure to (–)-anti-DB[a,l]PDE
Analysis of DNA adducts formed in HDF after treatment with increasing concentrations of (–)-anti-DB[a,l]PDE by 33P-post-labeling and subsequent HPLC separation always revealed a similar adduct pattern, as shown in the HPLC profiles obtained 42 h after treatment (Figure 5Go). The total amount of DNA adducts reached a maximal level of 219 pmol adducts/mg DNA 12 h after treatment with 0.07 µM (–)-anti-DB[a,l]PDE. The levels of adducts were 110 pmol adducts/mg DNA for cells treated with 0.028 µM and 0.9 pmol adducts/mg DNA for cells treated with 0.01 µM (–)-anti-DB[a,l]PDE.



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Fig. 5. Radiolabeled DNA adducts separated by HPLC on a C18 cartridge and measured with an on-line radioisotope flow detector. Buffer A, 0.1 M NH4H2PO4, pH 5.5; solvent B, 10% acetonitrile, 90% methanol; elution gradient, 20–49% B over 60 min, 49–65% B over 60 min. Peaks that elute with rentention times of 80–100 min are (-)-anti-DB[a,l]PDE–DNA adducts.

 
p53 and p21WAF1 response in asynchronous HDF after exposure to (–)-anti-DB[a,l]PDE
Western blot analysis of p53 and p21WAF1 protein levels in asynchronous HDF after treatment with varying concentrations of (–)-anti-DB[a,l]PDE are shown in Figure 6Go. An increase in the levels of both p53 and p21WAF1 was observed 24 h after treatment (Figure 6Go). The doublet band recognized by Ab-2 may represent different phosphorylation states of p53 (36).



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Fig. 6. Western blot analyses of lysates of HDF. Detection of p53 and p21WAF1 proteins by immunoblotting was performed as described in Materials and methods. A431 (human squamous cell line) protein was included on the blot as a postive control for p53 protein (32). Asynchronous HDF 24 h after treatment with 0.014 (lane 1), 0.028 (lane 2) and 0.07 µM (-)-anti-DB[a,l]PDE (lane 3), respectively. S, solvent (dimethyl sulfoxide)-treated control HDF.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
Cells expressing wild-type p53 have been shown to arrest in the G1 phase of the cell cycle upon exposure to DNA damaging agents (2,12,13) to give cells a longer time period for DNA repair. Previous studies in asynchronous human MCF-7 cells demonstrated that PAH–DNA adducts escape cell cycle arrest and cells with DNA damage accumulate in S phase (3740). In the present study the effect of PAH–DNA adducts on the cell cycle was investigated in non-transformed HDF that have an intact cell cycle regulatory machinery and are amenable to synchronization in G0 phase by contact inhibition (41), unlike the carcinoma cell line MCF-7.

The results obtained after exposure of synchronized HDF to increasing concentrations of (–)-anti-DB[a,l]PDE, an ultimately mutagenic and electrophilically reactive intermediate of the potent carcinogen DB[a,l]P, revealed growth arrest in the G1 phase of the cell cycle (Figures 3 and 4GoGo). A low dose of (–)-anti-DB[a,l]PDE (0.014 µM) did not arrest a significant proportion of cells in G1 phase compared with the control at 1, 12, 24 and 42 h after treatment (Figure 4Go). Thus dose-dependent G1 arrest was observed in HDF treated with (–)-anti-DB[a,l]PDE. This arrest in the G1 phase of the cell cycle at higher doses of (–)-anti-DB[a,l]PDE correlated well with the levels of DNA binding determined in these cells. The maximum DNA binding level was 219 pmol adducts/mg DNA after 12 h in HDF treated with 0.07 µM (–)-anti-DB[a,l]PDE, compared with 0.9 pmol adducts/mg DNA in cells treated with 0.014 µM (–)-anti-DB[a,l]PDE, also demonstrating concentration–dependent PAH–DNA adduct formation in HDF.

These results indicate that the cellular response of non-transformed human fibroblasts to PAH–DNA adduct formation results in induction of a significant cell cycle arrest in G1 phase and, therefore, the DNA damage-induced response of these cells fulfills all requirements for subsequent repair processes that may remove the DNA adducts prior to DNA replication. These findings are in apparent contrast to the observation of Dipple and co-workers in human breast carcinoma MCF-7 cells that PAH–DNA adducts could evade cellular defense mechanisms by escaping regulated cell cycle arrest (3740). This discrepancy could possibly result from using different cell types that may respond in a different manner to PAH-induced DNA damage. Further studies are needed to clarify the mechanisms underlying the different cellular responses of the two human cell lines. Using HDF the present study provides evidence that formation of a critical threshold level of PAH–DNA adducts is required before a cellular response occurs. This interesting aspect of PAH carcinogenesis in humans implies that low doses of the carcinogen may induce a low level of mutation while the cells escape cellular arrest and thus lack sufficient time for DNA repair processes to remove the damage prior to DNA replication. Consequently, even low levels of PAH–DNA adducts could generate mutations in the genome and, possibly, also within critical genes that may lead to the generation of cancer cells. Dipple and co-workers (3740) published this concept of `stealth carcinogens' with respect to the effects of carcinogenic PAHs. However, in the case of PAH-induced arrest in G1 phase concomitant with p53 stabilization at higher DNA adduct levels, the capacity and fidelity of cellular DNA repair processes may play a dominant role for protection against DNA damage-induced mutations. Bulky PAH adducts can be removed from DNA by nucleotide excision repair (NER) and in an in vitro study it was found that differences in the structure and configuration of PAH–DNA adducts are critical parameters for the efficiency of their removal from the DNA by NER (41). Recently Lloyd and Hanawalt (43) reported that efficient global NER of BPDE adducts is p53-dependent in human fibroblasts and that the requirement for p53 in global NER of BPDE adducts is dependent on the level of adducts being repaired. Thus the increase in repair is related to the initial basal levels of p53 protein rather than a damage-dependent increase because in repair-proficient p53-expressing cells the removal of BPDE adducts was almost complete before p53 protein levels were elevated (43).

B[a]P has been found to increase p53 protein levels in vivo (10,16) and in cell cultures (9,17). B[a]P- and BPDE-mediated recruitment of p53 is mainly due to an increase in protein stability (9,17,37,44) and a transcriptionally stimulated expression through induction of NF-{kappa}B (45). Our laboratory demonstrated previously that the level of DNA adducts formed by DB[a,l]PDE determined the effect on the proteins p53 and p21WAF1 in human MCF-7 cells (18,19). In the present study a dose as low as 0.014 µM (–)-anti-DB[a,l]PDE caused a detectable increase in the levels of p53 and p21WAF1 proteins in asynchronous HDF and increasing concentrations of (–)-anti DB[a,l]PDE resulted in further increases in the levels of both p53 and p21WAF1 24 h after treatment. The dose–response effect observed in asynchronous non-transformed HDF after treatment with (–)-anti-DB[a,l]PDE parallels the results obtained in the asynchronous carcinoma cell line MCF-7 (18,19). A substantial increase in p53 and p21WAF1 protein levels was observed in the carcinoma cell line MCF-7 at concentrations >0.01 µM (–)-anti-DB[a,l]PDE, accompanied by induced DB[a,l]PDE–DNA adduct levels >20 pmol adducts/mg DNA, while at lower DB[a,l]PDE–DNA adduct levels no detectable increase in either of these proteins was detected (18,19). Further studies of p53 and p21WAF1 proteins in synchronous HDF after treatment with (–)-anti-DB[a,l]PDE are in progress and may aid in understanding the possible regulation of these proteins and their dose dependency on the carcinogen.

Experiments are underway to examine how specific types of PAH–DNA adducts affect p53 and p21WAF1 protein levels and how they cause cell cycle arrest in human cells in culture. This would enable the determination of whether deoxyadenosine adducts differ from deoxyguanosine adducts in their ability to affect p53 and p21WAF1 protein levels and induce cell cycle arrest. One potential reason for differences in the carcinogenic potency of different PAHs could be the ability of their corresponding DNA adducts to affect p53 and p21WAF1 protein levels and thus induce cell cycle arrest. This may also help to understand why PAHs that form high proportions of deoxyadenosine adducts are more potent carcinogens than those which form mainly deoxyguanosine adducts in DNA (21).


    Notes
 
5 To whom correspondence should be addressed Email: william.baird{at}orst.edu Back


    Acknowledgments
 
This research work was supported by grants CA40228 (W.M.B.), CA72987 (J.C.P.) and CA81518 (J.C.P.) from the National Cancer Institutes, DHHS. We thank the Environmental Health Sciences Center (EHSC) at Oregon State University for use of the flow cytometer facility, which is part of the Cell and Tissue Analysis Facilities Service Core of the EHSC, which is supported by NIEHS Center Grant ES00210.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 8, 2000; revised October 16, 2000; accepted October 20, 2000.