of BPDE-induced p53 accumulation by TPA is associated with a decrease
in stability and phosphorylation of p53 and downregulation of NFκB
activation: role of p38 MAP kinase Abstract
DNA damage caused by benzo[a]pyrene (B[a]P) or other polynuclear hydrocarbons (PAHs) induce p53 protein as a protective measure to eliminate the possibility of mutagenic fixation of the DNA damage. 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits p53 response induced by B[a]P and other DNA-damaging agents and may cause tumor promotion. The molecular mechanism of attenuation of B[a]P-induced p53 response by TPA is not known. We investigated the effect of TPA on p53 response in (±)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)-treated mouse epidermal JB6(P+) Cl 41 cells. BPDE treatment induced p53 accumulation which was attenuated significantly by TPA. Cells treated with BPDE and TPA showed increased ratio of Mdm2 to p53 proteins in p53 immunoprecipitate and decreased p53 life span compared to BPDE-treated cells indicating p53 destabilization by TPA. TPA also inhibited BPDE-induced p53 phosphorylation at serine15. Activation of both ERKs and p38 MAPK by BPDE and attenuation of BPDE-induced p53 accumulation by U0126 or SB202190, specific inhibitor of MEK1/2 or p38 MAPK, indicate the role of ERKs and p38 MAPK in p53 accumulation. Interestingly, TPA potentiated BPDE-induced activation of ERKs whereas p38 MAPK activation was significantly inhibited by TPA, suggesting that inhibition of p38 MAPK is involved in p53 attenuation by TPA. Furthermore, SB202190 treatment caused decreased p53 stability and inhibition of phosphorylation of p53 at serine15 in BPDE-treated cells. We also observed that TPA or SB202190 attenuated BPDE-induced nuclear factor kappa B (NFκB) activation in JB6 Cl 41 cells harboring NFκB reporter plasmid. To our knowledge this is the first report that TPA inhibits chemical carcinogen-induced NFκB activation. Interference of TPA with BPDE-induced NFκB activation implicates abrogation of p53 function which has been discussed. Overall, our data suggest that abrogation of BPDE-induced p53 response and of NFκB activation by TPA is mediated by impairment of the signaling pathway involving p38 MAPK. Article
Many polynuclear aromatic hydrocarbons (PAHs) including benzo[a]pyrene (B[a]P) are well-known carcinogens and a considerable amount of data supports the role of these compounds in the induction of carcinogenesis (1,2). The cytochrome P-450-dependent monooxygenase system metabolically activates B[a]P preferentially to (+)-anti-B[a]P-7,8-diol-9,10-epoxide [(+)-anti-BPDE)], a reactive electrophile which binds to cellular DNA predominantly at the N2 position of deoxyguanosine (dG) and is implicated as the ultimate carcinogenic metabolite of B[a]P (3). Although B[a]P possesses carcinogenic potential, the tumorigenicity of B[a]P is greatly enhanced by the presence of tumor promoters (4,5). There is increasing evidence that DNA damage caused by B[a]P or other PAHs induces p53 levels in a number of animal or cell systems (6–9). Inhibition of p53 induction following DNA damage interferes with p53-mediated protective functions, which may lead to carcinogenesis.
Attenuation of DNA damage-induced p53 response by tumor promoters involves two fundamental events including inhibition of p53 accumulation and activation. It is observed that DNA damage-induced p53 accumulation is due mainly to an increase in p53 protein stability rather than to an increase in steady-state p53 mRNA levels (10,11). Stability and activation of p53 protein are mainly regulated by its interaction with mouse double minute 2 (Mdm2) oncoprotein and post-translational modifications (12–16). In normal, non-stressed cells, p53 has a short half-life of 15–20 min (17) and is rapidly degraded by ubiquitin-dependent proteolysis (18,19). The key mediator of p53 protein stability, Mdm2 protein, binds to the transactivation domain of p53 (amino acids 20–40) and functions as an E3 ubiquitin ligase, targeting p53 for ubiquitin-mediated proteolysis (12,13). The interaction of p53 with Mdm2 depends on the phosphorylation status of both p53 and Mdm2 proteins (20,21). p53 is known to be phosphorylated in vitro or in vivo by several kinases e.g. ATM, ATR, Chk1, Chk2, DNA-PK at several sites within its N- and C-terminal domains (14–16). Many of these kinase-mediated modifications are inducible upon DNA damage. Amongst the multiple modification sites on p53 molecule, it is observed that phosphorylations at Ser15 and Ser20 play an important role in p53 accumulation and function (20–23). It is also observed among other protein kinases that have been shown to phosphorylate p53 include mitogen-activated protein kinases (MAPK) (24–27). DNA damage is known to activate the MAPK pathway (28,29) and the dysregulation of MAPK signaling in human cancer is well documented (30). Stabilization and activation of p53 in response to DNA damage are known to be mediated by extracellular signal related kinase (ERK) 1 or 2 (24–26,31) and p38 MAPK (26–28,32), which phosphorylate p53 at several sites.
The biological significance of stabilization/activation of p53 through kinase-mediated phosphorylation relates to the transcriptional activating function of p53 which elicits cell cycle arrest and apoptosis (33). It is well known that p53-dependent cell cycle arrest requires transactivation of p21Waf1, which inhibits the cyclin-dependent kinases (CDK) (34). As candidates to mediate p53-dependent apoptosis, several p53 target genes including Bax and PIG genes have been suggested (35,36). Recently it has been observed that the induction of p53 causes activation of nuclear factor kappa B (NFκB) that correlates with the ability of p53 to induce apoptosis (37). Inhibition or loss of NFκB activity abrogated p53-induced apoptosis, indicating that NFκB is essential in p53-mediated cell death.
Abrogation of DNA damage-induced p53 stabilization/activation associated with the loss of p53 function may lead to tumor promotion. TPA (12-O-tetradecanoylphorbol-13-acetate) and other tumor promoters are known to inhibit p53 induction in vivo in mouse skin and in vitro in other mammalian cells in response to DNA-damage caused by B[a]P or other DNA damaging agents (38–42). On the contrary, it was observed that the tumor promoter phorbol ester alone induced p53 transcriptional activity and activation of NFκB (43–46). Although treatment of cells individually with either B[a]P or TPA causes activation of p53, the attenuating effect of TPA on BPDE-induced p53 response both in vitro and in vivo is very interesting. The mechanism of attenuation of B[a]P-induced p53 response by TPA or other tumor promoters has not been studied. In this study, we investigated the mechanism of attenuation of (±)-anti-benzo[a]pyvene-7,8-diol-9(BPDE)-induced p53 response by TPA in promotion-sensitive JB6 mouse epidermal cells (Cl 41) which are widely used for studies of the role of signal transduction pathways in tumor promotion (47–50). Here we demonstrate that attenuation of BPDE-induced p53 response by TPA is associated with a decrease in p53 stability and increased binding of Mdm2 with p53, and that TPA mediates this effect by interfering with p38 MAPK not ERKs, with concomitant inhibition of p53 phosphorylation at serine15 residue. We also showed that TPA severely interferes with BPDE-induced NFκB activation, a downstream target of p53 implicated in the apoptotic function of p53, and that p38 MAPK may have a role in this regard. To our knowledge this is the first report that TPA, a tumor promoter, inhibits chemical carcinogen-induced NFκB activation.
Materials and methods
Cells and reagents
Mouse epidermal JB6 Cl 41 (Cl 41) cells were obtained from American Type Culture Collection (ATCC, VA). The Cl 41 cells stably transfected with firefly luciferase reporter gene driven by a minimal NFκB-responsive region was a gift from Dr Nancy Colburn (51). (±)-anti-BPDE was purchased from the NCI Chemical Carcinogen Reference Standard Repository (Kansas, Missouri); Oncogene Research Product (San Diego, CA); Promega kit (Madison, WI). Modified Eagle's Medium (MEM), fetal calf serum (FCS), l-glutamine and trypsin-EDTA were purchased from Invitrogen Life Technologies (CA). Recombinant Elk-1 and ATF-2 fusion proteins, U0126 (MEK1/2 inhibitor) and primary antibodies against phospho-ATF-2 (Thr71), phospho-Elk-1 (Ser383), phospho-p53 (Ser15), p44/42 MAPK, phospho-p44/42 MAPK (Thr202/Tyr204), p38 MAPK and phospho-p38 MAPK (Thr180/Tyr182) were purchased from Cell Signaling (MA); monoclonal mouse anti-p53 and anti-Mdm2 (AB-3) antibodies were from Oncogene Rersearch Product (CA); polyclonal rabbit anti-p53 (FL-393) antibody and protein A/G PLUS-agarose were from Santa Cruz (CA); anti-rabbit and anti-mouse IgG conjugated with horseradish peroxidase were obtained from Sigma (MO); and SB202190 was from Calbiochem (CA). All other chemicals were of analytical grade.
Cl 41 cells were cultured as monolayers at 37°C in an atmosphere of 5% CO2 using MEM containing 5% FCS, 2 mM l-glutamine, 10 mM sodium pyruvate and penicillin/streptomycin (50 μg/ml each). The cell cultures were checked on a routine basis for Mycoplasma contamination by the Gibco Mycotect.
Cell treatment and preparation of whole cell extract
The cells in mid-log growth were treated with 1 μM (±)-anti-BPDE (BPDE) dissolved in DMSO (0.05–00.1% of the culture volume) for 90 min in serum-free medium and further incubated in 5% serum for 16 h (unless mentioned otherwise) in the absence of BPDE. For treatment with TPA, the cells in mid-log growth were treated with BPDE first for 90 min followed by treatment with 100 nM TPA for 1 h in serum-free medium and then further incubated in 5% serum. The cells, 16 h after 90 min of BPDE treatment, were washed three times with ice cold PBS, scraped gently and lysed in 100 μl of lysis buffer A consisting of 1% Triton X-100, 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 10 mM EDTA, 1 mM sodium β-glycerophosphate, 1 mM Na3VO4, 10 μg/ml pepstatin, 1 mM phenylmethanesulfonyl fluoride, 5 μg/ml leupeptin and 100 μg/ml aprotinin (Sigma). The supernatant collected after centrifugation (14 000 r.p.m. for 20 min at 4°C) was either used immediately or flash-frozen in liquid nitrogen and stored at −86°C for subsequent analyses.
Western blot and immunoprecipitation
Untreated cells and cells treated with either BPDE alone or BPDE and TPA were lysed in lysis buffer A, 16 h after BPDE treatment as described before, and the protein in the lysate was fractionated by SDS–PAGE and transferred to Immobilon P filters (Millipore). The membrane was blocked for 1 h with TBS containing 5% non-fat dry milk, 0.1% Tween 20 and then incubated for 1 h with the respective primary antibodies in TBS containing 5% non-fat dry milk. The antibodies used were FL-393 (Santa Cruz), Ab-3 (Oncogene Research Product) and anti-phospho-p53 (Ser15) (Cell Signaling) for p53, Mdm2 and p53 phosphorylated at serine15, respectively. Bound primary antibody was detected by incubating for 1 h with horseradish peroxidase-conjugated secondary antibody. The proteins were visualized by enhanced chemiluminescence using Amersham's ECL western blotting detection reagents (Amersham Biosciences, NJ).
The p53 bound Mdm2 protein level was determined by p53 co-immunoprecipitation assay. For the co-immunoprecipitation assay cells were lysed in lysis buffer A. Cell extract (300 μg of protein) was incubated with 3 μg of monoclonal mouse anti-p53 antibody overnight at 4°C with gentle rocking and then 20 μl (packed volume) protein A/G plus agarose beads was added with gentle rocking for 4 h at 4°C. The beads were washed extensively with lysis buffer and the pellet was re-suspended in 20 μl of 2× SDS buffer followed by SDS–PAGE fractionation. The levels of p53 and Mdm2 proteins in the immunoprecipitates were selectively measured by western immunoblotting using polyclonal rabbit anti-p53 and anti-Mdm2 (Ab-3) antibodies, respectively, and chemiluminiscent detection system.
Phosphorylation and kinase assay of ERKs and p38 MAPK
To determine the phosphorylation of ERKs and p38 MAPK, cells grown as monolayer (70–80% confluent) in 100 mm culture dishes were treated either with BPDE (90 min) alone or with BPDE (90 min) first and then with 100 nM TPA (1 h). Cells were lysed 4 h after BPDE treatment by adding 0.4 ml 1× SDS sample buffer in the dish and were scraped into microfuge tubes followed by brief probe sonication to sheer the DNA. Phosphorylation of ERKs and p38 MAPK in the respective cell extract was detected by western immunoblotting using specific anti-phospho antibody.
Kinase assays of ERKs and p38 MAPK were carried out as described by others (25). Briefly, the treated and untreated cells were harvested in 250 μl of lysis buffer. The cells were disrupted by repeated aspiration through a 21-gauge needle and then centrifuged at 15 000× g for 5 min at 4°C, and then the supernatant was used for immunoprecipitation. The supernatant fraction (300 μg protein) was incubated with 3 μg of specific ERKs or p38 MAPK antibody with gentle rocking at 4°C overnight. The immunocomplex was captured by adding 50 μl (20 μl packed beads) of washed Protein A/G plus agarose bead slurry by gentle rocking at 4°C for 2 h. The beads were collected by centrifugation and were washed 3 times with 500 μl ice-cold cell lysis buffer and then twice with 500 μl kinase buffer [25 mM Tris–HCl (pH 7.5), 5 mM beta-glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4 and 10 mM MgCl2]. The kinase reaction was carried out in the presence of 200 μM ATP in 20 μl kinase buffer at 30°C for 30 min using 2 μg of Elk-1 or ATF-2 as substrate for ERKs and p38 MAPK respectively. The reaction was terminated by adding 20 μl 3× SDS Sample Buffer and the phosphorylated proteins were detected by immunoblotting using phospho-specific antibody of the enzyme substrate Elk-1 or ATF-2.
NFκB activation assay
JB6(P+) Cl 41 cells harboring NFκB-responsive luciferase reporter gene were first selected in 5% serum-containing MEM in presence of 200 μg/ml G418. Cells grown at 70–75% confluence were treated with either BPDE or BPDE followed by TPA and were harvested 16 h after the BPDE treatment. Determination of luciferase activity in the respective cell extract was carried out according to Promega protocol. Briefly, the cells were lysed in lysis buffer (supplied in the kit), freeze-thawed and then centrifuged briefly at 14 000 r.p.m. An aliquot of 20 μl of the supernatant was mixed with 100 μl of the luciferase assay reagent (Promega kit) and the light intensity was measured by a Luminometer with a 2 s measurement delay and 10 s measurement read. NFκB activity in the respective cell extract was expressed as light units (LU) per mg protein.
Protein content in the cell extract was determined by using BCA reagent according to Pierce protocol.
Effect of TPA on p53 accumulation and Mdm2 binding to p53 in response to BPDE
Degradation of p53 is facilitated by binding of Mdm2 to p53 at the N-terminus (amino acids 19–26) (12,13). In order to assess the effect of BPDE on p53 and p53-bound Mdm2 protein levels, Cl 41 cells were treated with BPDE and the level of p53 in the cell extract was determined by western immunobloting (Figure 1A). The level of p53-bound Mdm2 protein was determined by co-immunoprecipitation with p53 antibody followed by western blotting of the immunoprecipitate using the respective antibodies (Figure 1B). A dose-dependent (0–1.0 μM) increase in the induction of p53 protein with BPDE was observed (Figure 1A, lanes 1, 2 and 4–6). The p53-bound Mdm2 protein level also increased in cells treated with 1.0 μM BPDE compared with untreated cells (Figure 1B, lane 2).
We examined whether TPA interferes with the BPDE-induced p53 accumulation which may abrogate p53-mediated protective functions in response to DNA damage. BPDE-induced p53 accumulation was significantly inhibited (63% inhibition) by TPA treatment (Figure 1A, lane 3).
A significantly higher ratio of Mdm2 to p53 proteins (3.2–1.0) in p53 immunoprecipitate from cells treated with BPDE and TPA compared with the ratio in the p53 immunoprecipitate from cells treated with BPDE alone (0.92–1.0) was observed (Figure 1B). Alpha Inotech software was used to quantify the protein band density in all western experiments. The Mdm2 protein level is under complex regulation in stressed cells. p53 protein which trans-activates Mdm2 gene is also known to be downregulated at protein level by Mdm2 through Mdm2-mediated ubiquitination and proteolytic degradation (12,13). We observed that the downregulation of BPDE-induced p53 accumulation by TPA is not associated with downregulation of the p53-bound Mdm2 protein level; rather an increased ratio of Mdm2 to p53 protein was observed in TPA treated cells. The data possibly indicate that TPA inhibits BPDE-induced p53 accumulation by promoting Mdm2-mediated destabilization of p53.
Interference of TPA with p53 stability
We studied the effect of TPA on p53 stability in BPDE-treated Cl 41 cells. The lifetime (stability) of p53 is determined according to the procedure described before (52). Cells were treated either with BPDE and then with TPA or with BPDE alone. Before harvesting cycloheximide (protein synthesis inhibitor) was added in the medium and the cells were harvested at different time points (0–1 h) and analyzed for the p53 protein levels by western immunoblotting (Figure 2). Cells treated with BPDE followed by TPA had significantly less proportion of p53 protein remaining 1 h after cycloheximide treatment (9.3% remaining) compared with cells treated with BPDE alone (57% remaining) (Figure 2, panels B and A). The data suggest that TPA downregulates p53 protein by decreasing its stability.
TPA inhibits p38 MAPK and p38 MAPK-inhibition attenuates BPDE-induced p53 accumulation, stabilization and phosphorylation.
Downregulation of BPDE-induced p53 accumulation by TPA prompted us to examine the involvement of the signaling pathway(s) which mediates the effect of TPA. We observed that cells treated with U0126, a specific inhibitor of mitogen activated protein kinase kinase (MEK or MAPKK) or SB202190, a specific inhibitor of p38 MAPK caused attenuation of BPDE-induced p53 accumulation (Figure 3A). SB202190 (2 μM) and U0126 (10 μM) caused 60% and 40% attenuation of BPDE-induced p53 accumulation, respectively. We further examined whether BPDE induces activation of ERKs and p38 MAPK, and whether TPA has an effect on the activation of ERKs and p38 MAPK in BPDE-treated cells. BPDE-treated cells showed increased level of phosphorylation of both the ERKs and p38 MAPK which corresponds with the increased activation of both kinases as evidenced by increased phosphorylation of their substrates Elk-1 and ATF-2, respectively (Figure 3B). Interestingly, TPA treatment caused potentiation of BPDE-induced phosphorylation and activation of ERKs whereas BPDE-induced phosphorylation and activation of p38 MAPK were significantly downregulated by TPA (Figure 3B). It is also observed that cells treated with U0126 (10 μM) and SB202190 (2 μM) at concentrations which inhibited p53 accumulation also inhibited BPDE-induced phosphorylation and activation of ERKs and p38 MAPK, respectively. These results indicate that although p53 accumulation (possibly stabilization) in response to BPDE involves activation of ERKs and p38 MAPK, TPA attenuates BPDE-induced p53 accumulation by interfering with p38 MAPK and not with ERKs. We also observed that p53 stability is reduced to a greater extent in cells treated with BPDE and SB202190 (Figure 3C; lower panel) compared with cells treated with BPDE alone (Figure 3C; upper panel). BPDE-treated cells show a much higher level of p53 1 h after cycloheximide treatment (57% remaining) compared with the cells treated with BPDE and SB202190 (15% remaining).
Post-translational modifications of p53 regulate its stability and activation (14–16). DNA damage-induced phosphorylation of p53 at Ser15/20 is important for p53 stability and activity (20–23). We further investigated whether BPDE-induced p53 accumulation is associated with the phosphorylation of p53 at Ser15/20 residues and whether TPA treatment or inhibition of p38 MAPK has any effect on p53 phosphorylation in BPDE-treated cells. We determined phspho-(Ser15/20) p53 levels in extracts of treated and untreated cells using phspho-(Ser15/20) p53-specific antibodies and observed that BPDE upregulated Ser15 phosphorylation of p53 (Figure 3D) but not Ser20 phosphorylation (data not shown). TPA or SB202190 caused significant inhibition of BPDE-induced p53 phosphorylation at Ser15 residue (Figure 3D). Next, we examined whether p53 accumulation and phosphorylation are also induced by proteasomal inhibitor N-Acetyl-Leu-Leu-Nle-CHO (ALLN) (Calbiochem) in order to compare with BPDE-induced p53 accumulation and phosphorylation, and whether TPA can attenuate ALLN-induced p53 accumulation and phosphorylation, if they occurred. Treatment of cells with 30 μM ALLN for 16 h induced p53 accumulation but not p53 phosphorylation at Ser15 and, interestingly, TPA did not attenuate ALLN-induced p53 accumulation (Figure 3E). These results suggest that proteasome inhibition-mediated p53 stabilization is different from BPDE-induced p53 stabilization with respect to p53 phosphorylation and inability of TPA to attenuate ALLN-induced p53 accumulation may have implication that p53 phosphorylation at Ser15 has possibly a role in BPDE-induced p53 stabilization which is interfered by TPA.
Taken together the above results suggest a possible role of p38 MAPK acting upstream of p53 in a signal transduction pathway initiated by BPDE, and that TPA interferes with this pathway by inhibiting p38 MAPK activity.
TPA interferes with NFκB activation
DNA damage-induced p53 response triggers a variety of signaling pathways to elicit protective events like apoptosis and cell cycle arrest (33). Since we observed that TPA attenuates p53 response to BPDE, we further investigated which signaling event(s) downstream of p53 is (are) affected by TPA. p53-dependent cell cycle arrest in response to DNA damage is mediated by p21WAF1 which is a transactivation product of p53 tumor suppressor protein. We did not observe any effect of TPA on p21WAF1 expression in cells treated with BPDE (data not shown). p53-mediated activation of the transcription factor NFκB is involved in the induction of apoptosis (37). Since NFκB has an important role in the p53-mediated apoptotic signaling event and our data showed that TPA abrogates BPDE-induced p53 response, we were interested in examining whether TPA interferes with NFκB signaling in cells treated with BPDE. NFκB activation was determined by NFκB-responsive luciferase reporter assay as described in Materials and methods. BPDE treatment of Cl 41 reporter cells showed a dose- and time-dependent induction of NFκB activity (Figure 4A and B). Up to 4 h after BPDE treatment, no significant increase in NFκB activity was observed followed by a gradual increase in NFκB activity with time, reaching the maximum at 16 h (53-fold induction) and remaining at this level up to 24 h (Figure 4A). Gradual increase of NFκB activity was also observed with increasing doses of BPDE treatment (Figure 4B). Cells treated with TPA alone also showed increased NFκB activity (3-fold) (Figure 4C). Most interestingly, TPA caused a marked inhibition (72%) of BPDE-induced NFκB activity instead of the additive activation expected for the combined action of TPA and BPDE (Figure 4C). To our knowledge this is the first report that TPA being a tumor promoter inhibits NFκB activation in chemical carcinogen-damaged cells. Since it is observed that TPA inhibits BPDE-induced p38 MAPK activation, we examined whether inhibition of p38 MAPK has an effect on BPDE-induced NFκB activation. It is observed that SB202190 treatment caused dose-dependent inhibition of BPDE-induced NFκB activation (Figure 4C). Treatment of cells with 1 and 2 μM SB202190 caused 22 and 48% inhibition of BPDE-induced NFκB activation, respectively. These results indicate that downregulation of BPDE-induced NFκB activation by TPA is mediated in part by inhibition of p38 MAPK.
In this investigation, we examined the effect of TPA on BPDE-induced p53 response in mouse epidermal cells (JB6 Cl 41) in order to obtain an insight into the mechanism by which TPA, an established tumor promoter, exerts its tumor promoting effect. The promotion-sensitive JB6 Cl 41 cells represent a suitable cellular model to study the tumor promoting activity of various compounds (51). p53 protein plays a pivotal role as a protective agent in cellular response to various types of genomic damage including chemical carcinogens. So, any interference with DNA damage-induced p53 response will be detrimental to normal cellular function and may lead to tumorogenesis. A previous study demonstrated that TPA caused downregulation of B[a]P-induced p53 accumulation in mouse skin (39). Our present study confirms this finding in the cell culture system indicating that BPDE induces p53 accumulation in a dose-dependent manner in Cl 41 cells and that TPA significantly attenuates BPDE-induced p53 response.
The mechanism of attenuation of p53 response by TPA has not been studied. Understanding the mechanism(s) by which TPA interferes with the cellular protective response to DNA damage will help gain an insight into the mechanism of tumor promotion and thus develop therapeutic strategy to prevent cancer. Our data indicate that destabilization of BPDE-induced p53 at protein level is one of the mechanisms by which TPA attenuates p53 accumulation in response to BPDE. Since Mdm2 binding to p53 protein triggers ubiquitin-mediated proteolysis of p53 (12,13), our finding of an increased binding of the oncoprotein Mdm2 with p53 in cells treated with BPDE followed by TPA compared with the cells treated with BPDE alone possibly indicates an increased degradation of p53 protein resulting in the attenuation of p53 stability by TPA. Stabilization of p53 protein which is negatively regulated by Mdm2 protein and activation of p53 responsible for its downstream functions are under complex regulation in cells under stress. The most important of these regulatory events is the post-translational modification by phosphorylation at specific sites. Phosphorylation of p53 at serine15 and 20 located at trans-activation domain is critical for stability and activation of p53 (20–23). It has been demonstrated that mutation of either serine15 or serine20 to alanine prevents the full stabilization and activation of p53 (20,22,23). Recent studies have proposed that DNA damage-induced phosphorylation of p53 at Ser15 and/or Ser20 attenuates p53–Mdm2 interaction (20,21). We observed that BPDE treatment caused phosphorylation of p53 at serine15 residue but not at serine20 and that TPA inhibits BPDE-induced phosphorylation at serine15 of p53. This suggests that attenuation of p53 accumulation and inhibition of p53 phosphorylation at serine15 are two events associated with TPA effect on cells damaged by BPDE.
To understand the regulation of these two events, it is important to identify the kinase(s) which is the upstream regulator of both the BPDE-induced stabilization and phosphorylation of p53. Our observation of significant attenuation of BPDE-induced p53 accumulation in cells treated with U0126 (MEK inhibitor) or SB202190 (p38 MAPK inhibitor) indicates the possible role of ERKs and p38 MAPK in p53 accumulation. It has been observed previously that ERKs and p38 MAPK have roles in stabilization and activation of p53 though phosphorylation at several sites (24–28,31,32). Interestingly, our results show that treatment of cells with TPA downregulates BPDE-induced p38 activation whereas ERK activation is potentiated significantly suggesting that inhibition of p38 kinase activity mediates the effect of TPA on cellular p53 response to BPDE. This inference is supported by further observation that SB202190 attenuates not only p53 accumulation but also both the p53 stability and p53 phosphorylation at serine15 in BPDE-treated cells. The question remains regarding the possible mechanism of upregulation of ERKs and downregulation of p38 MAPK by TPA in BPDE-treated cells. TPA is an established protein kinase C (PKC) activator and the involvement of PKC in phosphorylation and activation of Raf, MEK and ERKs is well documented (46,53). PKCα is also known to activate MAP kinases including p38 MAPK (54). In agreement with these reports, our observation of upregulation of ERKs by TPA possibly indicates a role of PKC. But downregulation of p38 MAPK by TPA in BPDE-treated cells observed by us can not be explained by PKC's involvement as observed by others (54). Further studies are needed to decipher the signaling event/s which explains the downregulation of p38 MAPK by TPA in BPDE-treated cells.
Inhibitory effect of TPA on p53 accumulation, stability and phosphorylation (serine15) indicates abrogation of p53 function. One of the important functions of tumor suppressor p53 is the activation of apoptotic signaling pathway (33,37). Induction of DNA damage-induced apoptotic signaling pathway in Cl 41cells is associated with p53 phosphorylation at serine15 (27,55). Regarding the involvement of signaling molecules which mediate the downstream apoptotic function of active p53, it has been reported that induction of p53 causes activation of NFκB that correlates with the ability of p53 to induce apoptosis (37). We observed that BPDE treatment induced NFκB activity several-fold and that TPA treatment caused significant inhibition of BPDE-induced NFκB activation. To our knowledge, this is the first report that the tumor promoter TPA downregulates NFκB activation in response to chemical carcinogen-induced DNA damage.
The question arises regarding the significance of downregulation of NFκB activity with respect to the tumor-promoting function of TPA. It is known that NFκB has dual roles towards the apoptotic function. It is a transcription factor that can protect or contribute to apoptosis (56). Although the mechanisms underlying the dual nature of NFκB function is not well understood it is suggested that the p53-independent apoptotic activity of E2F-1 is associated with the inhibition of NFκB activation by death receptors such as the TNFR (57), whereas p53-dependent apoptosis is associated with NFκB activation (37). Our observation of the inhibitory effect of TPA on BPDE-induced p53 response and NFκB activation possibly indicates the anti-apoptotic effect of TPA in BPDE-damaged cells, which may eventually lead to the acquisition of tumorogenic potential by initiated cells. Previous investigators reported that activation of ERKs and p38 MAPK is involved in the regulation of NFκB activation (37,58,59). Our findings indicate that TPA inhibits BPDE-induced NFκB activation by interfering with activation of p38 MAPK and not ERKs. Overall our data suggest that abrogation of chemical carcinogen-induced p53 response and of NFκB activation by TPA is mediated by impairment of the signaling pathway involving p38 MAPK. Further studies are underway to understand the mechanism of inhibition of NFκB activity by TPA in BPDE-damaged cells.
This work was supported by National Institute of Environmental Health Sciences (NIEHS) Grant R15ES12401 (to J.J.M.).
Conflict of Interest Statement: None declared.