Introduction

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Lung cancer is one of the most prevalent cancers in the world, and its mortality rate is expected to remain very high for many years (1). Concurrently, the environmental air quality is deteriorating, and the number of smokers has increased. Epidemiologic studies over the past several decades have provided a considerable body of evidence linking lung cancer to a number of mutagens and carcinogens detected in both the environment and cigarette smoke.

Polycyclic aromatic hydrocarbon carcinogens (PAHs), such as benzo(a)pyrene (B[a]P) and 1-nitropyrene (1-NP), are major environmental pollutants present in automobile exhaust, cigarette smoke, various foods, and industrial wastes. Carcinogenic and mutagenic effects of PAHs have been well documented in human, rodent, and other mammalian cell systems (2, 3). Most PAHs require metabolic activation to vicinal bay-region or fjord-region dihydrodiol epoxides if they are to express their carcinogenic activity and, in general, the PAHs are among the more potent known experimental carcinogens (4, 5). Active metabolites bind covalently to DNA and cause DNA damage (4, 6).

The p53 gene is one of the most commonly mutated genes identified in various types of human tumors, and the results of numerous studies suggest that the inactivation or abnormality of p53 is a critical step leading to neoplastic transformation (7). It is important for maintaining the integrity of the genome (8). Loss of p53 functions thus results in an enhanced frequency of genomic rearrangements or genomic instability (9) and eliminates the growth-arrest response or apoptosis induced by DNA-damaging genotoxic insults (12). In accordance with this function, p53 is recruited in response to various DNA-damaging agents such as ultraviolet, -irradiation, and anticancer drugs (13, 16, 17), and functions as a transcription factor to induce expression of various cellular genes involved in cell-cycle control and apoptosis, such as p21, Bax, and Mdm2 (7). The increased cellular p53 protein levels exposed to various genotoxic agents are due mainly to an increase in p53 protein stability rather than an increase in steady-state p53 messenger RNA (mRNA) levels (12, 16).

Recently it has been reported that PAHs and their metabolites result in a rapid accumulation of p53 gene product in human and mouse cells (19, 20). We also found that p53 protein is expressed in human atypical bronchial epithelium (ABE) and that the expressed p53 protein is the product of wild-type p53 gene in most bronchial biopsy specimens, suggesting that the wild-type p53 protein expressed in ABE might have a protective function from lung carcinogenesis (21). However, the induced p53 was reported to be transcriptionally inactive (22, 23). It was also found that PAHs stabilized p53 protein without induction of p21 and resulted in the cells' delay in S phase (22, 23). Although B[a]P, a kind of PAH, was reported to result in cell-cycle arrest in the G1 phase, this effect was found to be p53-independent (21). This prompted us to investigate the functional activity of the p53 protein resulting from PAH treatment. The experiments presented here demonstrated that PAHs-induced wild-type p53 protein is transcriptionally active.

Although most studies have focused on the mechanisms of transcriptional regulation of p21, it has recently been shown that p21 expression can also be regulated at the level of protein stability (24). p21 has been found to be ubiquitinated in vivo (25), suggesting that p21 levels may be regulated by the ubiquitin-proteasome pathway. This system plays a key role in cell-cycle control by regulating a number of regulatory proteins (26). In the present study, we found that PAHs increased ubiquitination of p21 protein after the stabilization of p53 and the expression of p21.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Carcinogens

B[a]P, 1,6-dinitropyrene (1,6-DNP), and 1-NP were purchased from Sigma-Aldrich (St. Louis, MO). 1-NP was prepared as described previously (2), and the purity of the 1-NP was greater than 99.99%. All chemicals were dissolved in dimethyl sulfoxide immediately before use and were added directly to the cell-culture medium as ×1,000 stocks to give the desired final concentration.

Cell Culture

Four different human lung-cancer cell lines differing in their p53 status were used in the present study: A549 (wild-type p53), NCI H460 (wild-type p53), NCI H1299 (p53 deleted), and NCI H322 (p53 mutated). All cells were maintained in RPMI 1640 medium containing 5% fetal bovine serum (CC Laboratories, Cleveland, OH).

Western Blots and Immunoprecipitation

For Western blot analysis without prior immunoprecipitation, the cells, treated with carcinogens, were washed twice with phosphate-buffered saline (PBS), scraped off, and then centrifuged. The total cell extracts were prepared by suspending the cell pellets in RIPA buffer (50 mM Tris [pH 7.2], 150 mM NaCl, 1% Nonidet P-40 [NP-40], 1% sodium deoxycholate, and 0.05% sodium dodecyl sulfate [SDS]). Equal amounts of protein were denatured by heating to 95°C in SDS-reducing buffer and were separated by electrophoresis on 8% (for p53, Mdm2) or 12.5% (for p21) SDS-polyacrylamide gels, and the proteins were electrophoretically transferred to Hybond-ECL nitrocellulose membranes (Amersham Pharmacia Biotech, Uppsala, Sweden). The membranes were probed with monoclonal antibodies (mAbs) anti-p53 (Ab-6), anti-WAF1 (Ab-1), or anti-Mdm2 (Ab-1) (Oncogene Science, Cambridge, MA). Bound antibodies were detected using an ECL Western blotting analysis system (Amersham Pharmacia Biotech). For detection of p21-ubiquitin protein conjugate, cell lysates were prepared in TNE buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% NP-40, 1 mM ethylenediaminetetraacetic acid [EDTA], 0.25% gelatin, 0.02% sodium azide, 1 mM phenylmethylsulfonyl fluoride [PMSF], 10 µg/ml leupeptin, and 10 µg/ml aprotinin). Extracts were cleared by centrifugation at 10,000 × g for 10 min, and anti-p21 polyclonal antibody (Ab 5; Oncogene Science) was added to the supernatant and incubated for 2 h at 4°C. Immobilized Protein A Gel (Pierce, Rockford, IL) was added subsequently, and the mixture was incubated for 2 h at 4°C. The beads were washed twice with TNE buffer and twice with 10 mM Tris-HCl (pH 7.5)-0.1% NP-40. The immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis and transferred to Hybond-ECL nitrocellulose membrane. The membrane was probed with anti-p21 mAb (Ab-1; Oncogene Science), and then the membrane was stripped and reprobed with a polyclonal antiubiquitin antiserum (StressGen Biotechnologies Corp., Victoria, BC, Canada).

Northern Blot Analysis

Cells were grown for 12 h, then treated for 2, 4, 8, or 24 h with B[a]P (1 µM) and 1-NP (1 µM), respectively. Total RNAs (20 µg) from each treatment were denatured with formaldehyde-formamide and separated in a 1% agarose-formaldehyde gel. RNAs were then transferred to Hybond-N nylon transfer membrane (Amersham Pharmacia Biotech). The membrane was hybridized with [³²P]-labeled complementary DNA (cDNA) fragments for human p21, Mdm2, or Bax. The cDNA probes were labeled with [³²P]deoxycytidine triphosphate by random priming using the Megaprime DNA labeling system (Amersham Pharmacia Biotech), and purified through NICK spin columns (Amersham Pharmacia Biotech). A -actin cDNA probe was later used to rehybridize the same membrane.

Plasmids

Luciferase reporter plasmids controlled by the 2.4-kb p21 promoter with (WWP-Luc) or without (DM-Luc) the p53-binding site were kindly provided by Dr. Toshiyuki Sakai (Kyoto Prefectural University of Medicine, Kyoto, Japan) (27, 28). A pRL-SV40 plasmid, Renilla luciferase control reporter vector, was purchased from Promega (Madison, WI).

Transient Transfection and Luciferase Assay

For DNA transfection, cells were plated at a density of 5 × 10⁵ cells per 60 mm-diameter culture dish 24 h before transfection. The cells were transfected with 2 µg of either WWP-Luc or DM-Luc and 0.1 µg of pRL-SV40 by the Lipofectamine Reagent (Life Technologies, Inc., Rockville, MD) according to the manufacturer's instructions. At 20 h after transfection, the cells were treated for 8 or 24 h with 1-NP in a serum-free medium and then harvested to determine their luciferase activities. The luciferase activities of the cell lysates were measured with a luminometer (Lumat LB9501; Berthold, Pforzheim, Germany) using a Dual-luciferase reporter assay system (Promega). Both Renilla luciferase and luciferase were measured, and the Renilla values were used for normalization.

Preparation of Nuclear Extracts

For the isolation of nuclear extracts, all procedures were performed on ice. Nearly confluent monolayers of cells, which had been treated with PAHs or control medium for the appropriate time, were washed with PBS, harvested by scraping into 1 ml of PBS, and pelleted at 5,000 rpm for 5 min. The pellet was washed twice with PBS, and then suspended in 0.4 ml of lysis buffer (10 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid [Hepes] [pH 7.9], 10 mM KCl, 0.1 mM EDTA, 1.5 mM MgCl₂, 0.25% NP-40, 1 mM dithiothreitol [DTT], and 0.1 mM PMSF). The pellet was vortexed and centrifuged at 5,000 rpm for 2 min, and then resuspended in 60 µl of extract buffer (20 mM Hepes [pH 7.9], 420 mM NaCl, 0.1 mM EDTA, 1.5 mM MgCl₂, 25% glycerol, 1 mM DTT, and 0.5 mM PMSF and rocked for 30 min at 4°C. After centrifugation, the supernatant containing nuclear extract was removed, and the protein concentration was determined. The nuclear extracts were then stored at 70°C until further use.

Electrophoretic Mobility Shift Assay

Electrophoretic mobility shift assays were performed by incubating 10 µg of nuclear extract in 10 µl of binding buffer (10 mM Tris-HCl [pH 7.5], 50 mM NaCl, 0.5 mM DTT, 0.5 mM EDTA, 1 mM MgCl₂, 4% glycerol, and 0.25 mg/ml poly[dI-dC]) for 10 min at 4°C. Then 35 fmol of [³²P]-labeled oligonucleotide probe was added, and the reaction mixture was incubated for 30 min at room temperature. For reactions involving competitor oligonucleotide, the unlabeled competitor and the labeled probe were premixed before addition to the reaction mixture. For supershift assays, the reaction mixture minus the probe was incubated with 2 µl of specific antibody PAb421 (Oncogene Science) for 1 h at 4°C. The [³²P]-labeled oligonucleotide was then added and incubated for 30 min at room temperature. The samples were electrophoresed at 4°C for 2 h on a 4% nondenaturating polyacrylamide gel (30:1) in a Tris-Borate-EDTA buffer system. The dried gels were exposed to Kodak X-Omat film at 80°C.

  The specific probe that was used for binding, 5'-CCGGGCATGTCCGGGCATGTCCGGGCATGT-3', contains the high-affinity binding sequence identified by Halazonetis and colleagues (29), named by them as BC or BB.9. Complementary single-stranded oligonucleotides were annealed by incubation at 85°C for 5 min and then gradually brought to room temperature. The probe was end-labeled with [-³²P]adenosine triphosphate (Amersham Pharmacia Biotech) using T4-polynucleotide kinase (Takara, Kyoto, Japan), after which unincorporated nucleotides were removed with NICK spin columns.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

PAHs Increased p53 Accumulation

Western blot analysis of p53 protein after the treatment with either B[a]P or 1-NP indicated that they induced the wild-type p53 protein to a higher level in A549 cells. Treatment of the cells with B[a]P or 1-NP resulted in a p53 induction response that was apparent with doses as low as 0.1 µM. The p53 induction response was saturable at both 8-h (Figure 1A) and 24-h points (data not shown) from treatment with doses higher than 1 µM of B[a]P or 1-NP. Time-course experiments revealed that p53 protein begins to accumulate as early as 2 h and remains at elevated levels for at least 24 h after exposure (Figure 1B).

View larger version (46K): [in this window] [in a new window]   Figure 1.   Dose- and time-dependent accumulation of p53 protein in A549 cells treated with B[a]P or 1-NP. Exponentially growing cells were treated with the indicated doses of B[a]P or 1-NP for 8 h (A) or with 1 µM of B[a]P or 1-NP for the indicated times (B), and whole-cell extracts were analyzed by Western blotting using an anti-p53 mAb (Ab-6). The cells treated with vehicle only were used as controls. Results are representative of four separate experiments.

B[a]P-treated and 1-NP-treated NCI H460 cells carrying wild-type p53 displayed an identical response with A549 cells. Unexpectedly, protein levels of p53 induced by 1-NP were much higher than those induced by B[a]P at all time points. 1,6-DNP, another nitropyrene, induced p53 protein levels similar to those induced by B[a]P (data not shown). When NCI H322 cells (p53 mutated) were treated with B[a]P and 1-NP, no increase of p53 protein was observed (data not shown).

Expression of Mdm2, p21, and Bax mRNA Was PAHs- and p53-Dependent

It has been reported that PAHs stabilized p53 without induction of p21 protein level, suggesting that the induced p53 was transcriptionally inactive (22, 23). We therefore investigated the expression of p21 mRNA in A549 cells. In the cells treated with 1 µM B[a]P or 1-NP, a substantial increase in p21 mRNA expression was seen by 4 h, and the level remained high up to 24 h after the treatments (Figure 2). Expression of p21 mRNA in A549 cells treated with 1-NP was much higher than that in cells treated with B[a]P at all time points, which was in parallel with the increase in p53 protein as shown in Figure 1B. To further study the transcriptional activity of p53 induced by treatment with PAHs, mRNA expression of Mdm2 and Bax, two other p53 target genes, was also measured in the same cells treated with B[a]P and 1-NP, respectively. As shown in Figure 2, the expression patterns of Mdm2 and Bax mRNA were similar to that of p21 mRNA, indicating that those mRNA expressions were related to the activity of the accumulated p53 protein. PAHs dose-dependent expression of p21 mRNA was also confirmed in A549 cells (Figure 3A). Its expression patterns were also concordant with those of the p53 protein shown in Figure 1A. To exclude the possibility that the phenomena are restricted to A549 cells, the ability of PAHs-induced p53 to transactivate its target gene p21 was also investigated in NCI H460 cells. As shown in Figure 3B, either B[a]P or 1-NP induced p21 mRNA expression in a dose-dependent manner, which was perfectly consistent with the response in A549 cells.

View larger version (65K): [in this window] [in a new window]   Figure 2.   Effect of B[a]P and 1-NP on mRNA expression of p53 target genes p21 (top row), Mdm2 (second row), and Bax (third row) in A549 cells. After treatment with B[a]P (1 µM) or 1-NP (1 µM) for the indicated times, total cellular RNAs extracted from A549 cells were hybridized to human p21, Mdm2, or Bax cDNA probes; the same blot was rehybridized to a -actin probe (bottom row). The cells treated with vehicle only were used as controls. Results are representative of four separate experiments.

View larger version (55K): [in this window] [in a new window]   Figure 3.   Dose-dependent expression of p21 mRNA in A549 (A) and NCI H460 (B) cells treated with B[a]P or 1-NP. The cells were treated with the indicated doses of B[a]P or 1-NP for 8 h. Total cellular RNAs were extracted and hybridized to human p21 cDNA probe. The cells treated with vehicle only were used as controls. Ethidium bromide staining of the agarose gels is shown as a control for the amounts of RNA loaded in each lane (20 µg/ lane). The positions of 28S and 18S ribosomal RNAs are indicated. Results are representative of four separate experiments.

Because p21 may be induced in a p53-independent manner by DNA-damaging agents (30), we next investigated the p21 mRNA expression in NCI H1299 (p53 deleted) and NCI H322 (p53 mutated) cells in response to B[a]P and 1-NP. As shown in Figure 4, no apparent increase in p21 mRNA was observed in both NCI H322 and NCI H1299 cells after B[a]P and 1-NP treatments.

View larger version (49K): [in this window] [in a new window]   Figure 4.   Effects of B[a]P and 1-NP on p21 mRNA expression in NCI H322 (A) and NCI H1299 (B) cells. The cells were treated with 1 µM of B[a]P or 1-NP for the indicated times. Total cellular RNAs were extracted and hybridized to human p21 cDNA probe. The same blot was rehybridized to a -actin probe. The cells treated with vehicle only were used as controls. Results are representative of three separate experiments.

1-NP Activated p21 Promoter Activity in A549 Cells

The effect of 1-NP on the transcriptional activation of the p21 promoter and its p53 dependence was investigated using a set of reporter constructs. The luciferase gene was driven either by the intact p21 promoter (WWP-Luc) or by a truncated p21 promoter in which the major p53-binding element at 2.4 kb is missing (DM-Luc) (27, 28). Transcription from the wild-type p21 promoter was markedly induced by 1 µM of 1-NP in p53 wild-type A549 cells (Figure 5). However, when NCI H1299 (p53-deleted) cells were transfected with WWP-Luc and exposed to 1-NP, luciferase activity was not affected (data not shown). The p53 binding site was further demonstrated to be necessary for transcriptional activation from the p21 promoter by 1-NP, as no significant changes in transcription were detected from the truncated promoter (DM-Luc) in A549 cells treated with 1-NP (Figure 5). These results suggest that 1-NP- induced p53 is transcriptionally active and that 1-NP regulates p21 promoter activity through the p53 protein.

View larger version (17K): [in this window] [in a new window]   Figure 5.   Activation of p21 promoter by 1-NP in A549 cells. The cells were transfected with reporter constructs driven by the full-length p21 promoter (WWP-Luc) or a truncated promoter lacking the p53 binding site (DM-Luc). At 20 h after transfection, the cells were treated for an additional 8 or 24 h with 1 µM of 1-NP. Luciferase activity was normalized to the activity of cotransfected Renilla luciferase (pRL-SV40). The luciferase activity is expressed as the fold increase in induction relative to the control (CTL) treated with vehicle only. Results represent the average of three independent experiments.

1-NP Enhanced Sequence-Specific Binding Activity of the p53 Protein in A549 Cells

To test whether increased p53 protein in A549 cells treated with PAHs was correlated with increased p53- DNA binding activity, we studied the ability of the p53 protein to bind to the p53 consensus sequence. A549 cells were treated with 1-NP for 8 h, and then nuclear extracts were prepared. There was a significant increase in the p53-DNA binding activity in the nuclear extracts derived from 1-NP-treated A549 cells as compared with the untreated control cells (Figure 6). The p53-specific band was subject to competition with an excess of unlabeled self oligonucleotide. Supershift analysis using anti-p53 mAb further confirmed the binding of p53 protein to the consensus DNA-binding elements.

View larger version (51K): [in this window] [in a new window]   Figure 6.   Activation of p53 DNA-binding activity after 1-NP treatment. A549 cells were treated with either 1-NP (1 µM) or an equivalent volume of vehicle (control) for 8 h. Nuclear extracts were prepared and incubated with the [32P]-labeled oligonucleotide BC. Specificity of the oligonucleotide was confirmed by competition experiments with 3- or 30-fold excess of unlabeled BC oligonucleotide (lanes 3 and 4). Supershift assay was performed using mAb PAb421. Black arrow marks the position of the p53-specific band; open arrow, the position of the antibody supershifted band. Results are representative of three separate experiments.

PAHs-Induced p21 mRNA Did Not Result in Proportional p21 Protein Increase

Next, we investigated the protein levels of p21 in A549 cells treated with B[a]P and 1-NP. The same blot as that in Figure 1A was reprobed with the monoclonal anti-p21 antibody (Figure 7A). Strikingly, no significant increase in p21 protein levels was observed in the cells treated with those carcinogens in spite of the higher levels of mRNA expression induced by the treatments (compare with Figure 3A), suggesting the existence of post-transcriptional modification. Further, the same blot as that in Figure 1B was also reprobed with anti-p21. Except for the slight increase of p21 protein in the cells treated with 1-NP for 24 h (Figure 7B), no significant induction of p21 protein was found by the treatments at all other time points. To exclude the phenomena that may also occur in other p53 downstream target genes, the blots in Figure 1 were also reprobed with the monoclonal anti-Mdm2 antibody. As expected, Mdm2 protein levels increased in proportion to mRNA levels (Figure 7), which were also perfectly consistent with the p53 protein levels (compare with Figures 1 and 2).

View larger version (14K): [in this window] [in a new window]   Figure 7.   Mdm2 and p21 protein levels in A549 cells exposed to B[a]P or 1-NP. The same blot as in Figure 1A (A) or 1B (B) was reprobed with mAbs against Mdm2 and p21, respectively. The Mdm2 and p21 bands are indicated.

PAHs Induced Proteasome-Dependent Degradation of p21 Protein

To analyze the regulation of p21 expression by proteasome, we examined the effect of MG-132, a proteasome inhibitor, on p53 and p21 expression. First, the p21 mRNA level was examined after treatment with MG-132 for 8 h. The level of p21 mRNA was slightly increased after treatment with 25 µM of MG-132 (data not shown). Next, we examined the effect of MG-132 on p21 protein product. A significant increase in the level of p21 protein was observed in A549 cells exposed to MG-132 (Figure 8, lanes 2, 3, and 4 of lower panel). No significant increase was observed in the level of p21 protein in the cells exposed to 1-NP or B[a]P alone (Figure 8, lower panel, lanes 5 and 9). However, when the cells were treated with either 1-NP or B[a]P in the presence of MG-132, the p21 protein was markedly increased (Figure 8, lanes 6-8 and 10-12). To further understand the mechanism of p21 expression in A549 cells exposed to PAHs, we analyzed the effect of MG-132 on p53 protein expression. As shown in the upper panel of Figure 8, B[a]P, 1-NP, and MG-132 all increased the levels of p53 protein, among which the 1-NP-induced p53 protein level was much higher than those induced by either B[a]P or MG-132 (Figure 8, lanes 2-4, 5, and 9). However, in the same condition the protein level of p21 induced by 1-NP was less than that induced by MG-132 (Figure 8, lanes 2-4 and 9), suggesting the important role of MG-132 in regulation of the protein level of p21. When the cells were treated with B[a]P in combination with MG-132, both p53 and p21 protein levels were markedly increased (Figure 8, lanes 6-8). However, the increase in p21 protein levels was significantly larger than the increase in p53 protein levels. Although the p53 protein level was not changed in spite of treatment with both 1-NP and MG-132, p21 protein was still markedly increased (Figure 8, lanes 10-12). In addition, although p53 protein levels induced by B[a]P in combination with MG-132 were much lower than those induced by 1-NP treatment only, p21 protein levels induced by B[a]P in combination with MG-132 were much higher than those induced by 1-NP only (Figure 8, lanes 6-9). Lastly, to determine whether the degradation of p21 following 1-NP or B[a]P treatment was due to a stimulation of the ubiquitination of p21 in vivo, p21 protein from A549 cells treated with or without 1-NP, B[a]P, or MG-132 was immunoprecipitated with a p21 polyclonal antibody, and the immunoprecipitates were examined by Western blot analysis with antibodies against p21 or against ubiquitin (Figure 9). A ladder of bands whose sizes were consistent with the addition of one, two, or three ubiquitin molecules to p21 were recognized by the p21 mAb (Figure 9, right). These bands were significantly increased in the cells that had been treated with 1-NP or B[a]P. The same blot was stripped and reprobed with an antiubiquitin polyclonal antibody (Figure 9, left), and the same ladder of bands was detected. The protein bands recognized by both antibodies are denoted with asterisks in the figure and are ubiquitinated forms of p21, indicating that ubiquitination of p21 was enhanced by 1-NP or B[a]P treatment. Proteasome inhibitor treatment of A549 cells also resulted in the appearance of these bands. Because the treatment with PAHs for 8 h did not result in significant increase in p21 protein levels, cell extracts from A549 cells treated with 1-NP for 24 h were used as a positive control to be immunoprecipitated by p21 polyclonal antibody. As shown in the far right lane of Figure 9, the full-length, nonubiquitinated p21 protein in the immunoprecipitates from the extracts was recognized by the p21 mAb but not by the antiubiquitin antibody (data not shown).

View larger version (27K): [in this window] [in a new window]   Figure 8.   Effect of proteasome inhibitor on p53 and p21 protein expression. Exponentially growing cells were treated with the indicated doses of MG-132 with or without either B[a]P (1 µM) or 1-NP (1 µM) for 8 h, and whole-cell extracts were analyzed by Western blotting using mAbs against p53 or p21. The cells treated with vehicle only were used as controls. Results are representative of four separate experiments.

View larger version (39K): [in this window] [in a new window]   Figure 9.   Detection of ubiquitinated p21 in B[a]P-treated or 1-NP- treated cells. A549 cells were either untreated, treated with the proteasome inhibitor MG-132 (25 µM), or treated with B[a]P (1 µM) or 1-NP (1 µM) for 8 or 24 h. Cell lysates were prepared as described in MATERIALS AND METHODS. p21 was immunoprecipitated from the extracts using the p21 polyclonal antibody Ab5, and the immunoprecipitates were examined by Western blot analysis with the p21 mAb Ab1 (right). The blot was then stripped and reprobed with an antiubiquitin polyclonal antibody (left). The open arrow indicates the p21 band. Asterisks denote protein bands detected by both p21 and ubiquitin antibodies. Background bands are indicated by the filled arrows and are most likely due to detection of the antibody heavy chain (50 to 55 kD) and light chain (25 to 30 kD) used in the immunoprecipitation.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Accumulation of cellular p53 protein in response to PAHs and their metabolites has been demonstrated in several earlier reports (19, 20, 22, 23), but the transcriptional activity of the p53 protein resulting from the PAHs treatment and the induction of p53 downstream target genes, especially that of p21 gene expression, are still uncertain. In this study, we analyzed the effects of B[a]P and 1-NP on p53 protein accumulation and the transcriptional activity of the protein. We demonstrated the involvement of the ubiquitin-proteasome degradation pathway in post-transcriptional regulation of p21 expression.

DNA damage induced by B[a]P and its metabolites increased p53 protein in human and mouse cells (19, 20, 22, 23). p53 protein expression is correlated with B[a]P-DNA adducts (20, 31). However, the conflicting results of PAHs on p53 and p21 expression have been reported (19, 20, 22, 23, 32). Thus, we selected 1-NP and 1,6-DNP, two other kinds of PAHs besides B[a]P, to confirm the effect of PAHs on p53 protein accumulation and the expression of its downstream target genes. Both 1-NP and 1,3-DNP have been well reported to form DNA adduct in human cells (3). So it is reasonable that 1-NP and 1,6-DNP also accumulate p53 protein in human lung-cancer cells, although up to now, as we know, no research on p53 protein expression has been done using these two carcinogens. Surprisingly, 1-NP, whose mutagenicity to human cells is much less than both B[a]P and 1,6-DNP (5), increased p53 protein accumulation much more than either B[a]P or 1,6-DNP did at all time points. The mechanism by which 1-NP induced a dramatic accumulation of p53 protein is still unclear, but this may reflect a potential of the cells to eliminate or repair the damaged DNA that resulted from 1-NP treatment. Further, it provides us a good model to study the effects of PAHs on p53 and p53 downstream target genes.

Because transcriptional activity is one of the most important mechanisms by which p53 functions to arrest cells at the G1 phase, to induce apoptosis, and to repair DNA in response to DNA damage (7, 11, 33), it is important to elucidate the transcriptional activity of p53 protein. The ability of p53 protein induced by PAHs treatment to transactivate its target genes p21, Mdm2, and Bax was therefore investigated at the mRNA level in different cell types. We found that wild-type p53 protein levels induced by PAHs treatment were correlated directly to the mRNA levels of p21, Mdm2, and Bax, suggesting that the induced p53 was transcriptionally active. Although no apparent increase in p21 mRNA was observed in both NCI H1299 (p53-deleted) and NCI H322 (p53-mutated) cells after B[a]P and 1-NP treatments, the expression of p21 was revealed in a p53-dependent manner. These results were consistent with those derived from most DNA-damaging treatments (7, 24) but inconsistent with some reports obtained from B[a]P or its metabolite treatments (20, 22, 23). Khan and associates (22, 23) reported that a benzo[g]chrysene metabolite, a PAH carcinogen, stabilized p53 without induction of p21 protein. They therefore suggested that the induced p53 protein was transcriptionally inactive (22, 23). However, they did not check the expression of p21 in mRNA level, and, further, in their experiments p21 protein level was, in fact, increased after treatment for 21 h. This supports our results and those of Luch and colleagues (32), who found that the level of p21 protein did not exceed control values until 12 to 24 h or more after exposure to PAHs and their metabolites. Therefore, any cell-cycle arrest caused by increases in p21 in response to the PAHs- induced DNA damage may occur after replication of DNA containing appreciable levels of PAHs-DNA adducts. Similar prolonged p53-dependent G1 arrest and long-term induction of p21 were described in normal human fibroblasts treated with -radiation (34). Vaziri and Faller revealed that B[a]P-induced p53 did not change the expression of p21 in 3T3 fibroblasts (20). Although they did not find B[a]P-dependent changes in p21 transcript levels, Mdm2 mRNA level was clearly induced by B[a]P. Further, they checked the effect of B[a]P on p21 mRNA expression by exposure of Swiss 3T3 cells to B[a]P for only 12 h, which would be insufficient to affect the p21 mRNA level. In our experiments B[a]P did not affect the p21 mRNA level until 24 h exposure. This may be due to the weak effect of B[a]P on p53 expression.

Most studies have shown that induction of p21 is p53-dependent (24, 27, 35), whereas some studies have suggested the induction of p21 by a p53-independent pathway (30). Our results suggest that the high level of the wild-type p53 protein resulting from the PAHs treatment is responsible for induction of p21. 1-NP induced transcription from the p21 promoter only in wild-type p53 cells, and this required the major p53 binding site in the promoter, suggesting that 1-NP-induced p53 is transcriptionally active and that 1-NP regulates p21 promoter activity through the p53 protein. The gel-shift assay demonstrated that activation of p53-binding ability to p53 consensus sequence was obvious 8 h after 1-NP treatment, further confirming the transcriptional activity of the induced p53 protein.

Although PAHs treatment accumulated wild-type p53 protein in good correlation with the expression of p21, Bax, and Mdm2 mRNA, suggesting the transcriptional activity of the induced p53 protein, the increased p21 mRNA level did not result in proportional protein product. The discrepancy between the mRNA level and protein product level suggests that p21 expression is post-transcriptionally regulated. When A549 cells were treated with PAHs and MG-132 together, a high level of p21 protein product was detected. The data indicate that p21 mRNAs induced by PAHs treatment in A549 cells were translated efficiently, and the protein product remained intact with the addition of proteasome inhibitor. p21 mRNA levels were slightly increased in the cells after treatment with MG-132 for 8 h. In contrast, a large increase in the level of p21 protein was observed in the cells exposed to MG-132. Similar results have previously been reported (36, 37). Treatment of the cells with PAHs and MG-132 together resulted in the increase of both p21 and p53 protein levels; however, the increase in p21 protein levels was significantly larger than the increase in p53 protein levels. These results clearly suggest that the p21 product is degraded by the ubiquitin-proteasome system and that the regulation of translational initiation of p21 mRNA is not likely to be a major regulatory mechanism in this case.

Further detection of ubiquitinated p21 in A549 cells after treatment with proteasome inhibitor and PAHs supports the notion that the ubiquitin-proteasome pathway is involved in the regulation of p21 expression. Previous studies found that ubiquitinated p21 bands were observed in cells transfected with ubiquitin expression vector or treated with proteasome inhibitors (25, 38). Treatment with PAHs increased the level of ubiquitinated p21, suggesting the existence of mechanisms for activation of ubiquitin ligase activity or inhibiting deubiquitination by PAHs.

PAHs react with and damage cellular DNA. Exposure to these carcinogens does lead to increased levels of p53 protein. However, p21 protein, transactivated by the induced p53 protein, is degraded immediately after its induction. The degradation of the protein is enhanced by PAHs treatment and thus leads to the absence of a G1 arrest that allows PAHs to damage DNA without turning on the cells' "guardian of the genome" defense mechanism, presumably increasing the likelihood of malignant change because DNA replication continues on a damaged template. Although the mechanism of p21 protein change in response to PAHs treatment in the present study is different from the concept that PAHs can act as "stealth carcinogens" proposed by Khan and coworkers (22, 23), the result of the treatment allowing replication before p21 protein increase and cell-cycle arrest is similar, suggesting a possible contributor to the carcinogenic potency of the carcinogens.

In summary, the present study demonstrated that PAHs treatment led to a rapid and sustained increase in cellular p53 levels. The induced p53 protein transactivated the expression of p53 downstream target genes Mdm2, p21, and Bax. PAHs activated the p21 promoter in a p53-dependent manner and enhanced p53-binding ability to p53 consensus sequence, further confirming the transcriptional activity of the induced p53 protein. In addition, PAHs increased the ubiquitination of p21 protein. The reason B[a]P and its metabolites stabilize p53 protein without induction of p21 protein, as previously reported, may not be due to the transcriptional inactivity of the p53 but may be due to the increase of p21 ubiquitination.