Increase in wild-type p53 stability and transactivational activity by the chemopreventive agent apigenin in keratinocytes

Maralee McVean1, Hengyi Xiao3, Ken-ichi Isobe3 and Jill C. Pelling1,2,4

1 Department of Pathology and Laboratory Medicine and
2 Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA and
3 Department of Basic Gerontology National Institute for Longevity Sciences, 36-3 Gengo Morioka-Cho Obu, Aichi 474-8522, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Apigenin, a naturally occurring, non-mutagenic flavonoid, has been shown to inhibit UV-induced skin tumorigenesis in mice when topically applied. In this report we have used the mouse keratinocyte 308 cell line, which contains a wild-type p53 gene, to study the effect of apigenin treatment on p53 protein levels and the expression of its downstream partner, p21/waf1. Cells were treated with 70 µM apigenin for various times and levels of p53 and p21/waf1 protein were assessed by western blot analysis. The level of p53 protein was induced 27-fold after 4 h of apigenin treatment and levels remained elevated through 10 h of exposure. After 24 h of exposure to 70 µM apigenin, p53 protein levels returned to control levels. p21/waf1 protein levels increased ~1.5–2-fold after 4 h and remained elevated at 24 h. To investigate the mechanism of p53 protein accumulation, we compared the half-life of p53 protein in vehicle- and apigenin-treated cells. Cells were incubated for 4 h in the presence of apigenin, then cycloheximide was added to inhibit further protein synthesis and p53 protein levels were measured by western blot. The half-life of p53 protein was found to be increased an average of 8-fold in apigenin-treated cells compared with vehicle-treated cells (t1/2 = 131 min versus 16 min in apigenin- versus vehicle-treated cells, respectively). The mechanism of p53 protein stabilization is currently being investigated. To determine whether p53 was transcriptionally active, we also performed gel mobility shift assays and transient transfection studies using a luciferase plasmid under the control of the p21/waf1 promoter. Both p53 DNA-binding activity and transcriptional activation peaked after 24 h of exposure to apigenin. These studies suggest that apigenin may exert anti-tumorigenic activity by stimulating the p53–p21/waf1 response pathway.

Abbreviations: Cdk, cyclin-dependent kinase; CKII, casein kinase II; DMSO, dimethyl sulfoxide; JNK1, jun-amino-terminal kinase; PKC, protein kinase C; TLB, Tris lysis buffer.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Apigenin is a non-mutagenic flavonoid (1) displaying a variety of anti-tumor effects including stimulation of gap junctional and intercellular communication (2) and inhibition of mutagenesis (3), transformation (3,4), angiogenesis (5) and tumorigenesis (5,6). Topical application of apigenin in mice has been shown to decrease ornithine decarboxylase activity (7) and the number and size of tumors in the skin induced both by chemical carcinogens (7) and UV exposure (6). These studies suggest that apigenin may be a useful chemopreventive agent against skin cancers in humans.

Investigations into the mechanisms underlying the anti-tumorigenic properties of apigenin have shown that treatment can trigger numerous cellular events. Apigenin treatment resulted in G1 cell cycle arrest in synchronized human diploid fibroblasts (8) and G2/M arrest in rat neuronal cells (9) and mouse keratinocytes (10). Cell cycle checkpoints regulate passage through the cell cycle, causing arrest after DNA damage or cellular disruption. Arrest allows the cell time to repair damaged DNA and rectify alterations in gene expression. p53 has been shown to regulate passage through both G1 and G2/M (11,12). Overexpression of p53 inhibited cellular proliferation (13) and activation of p53 in response to DNA damage led to cell cycle arrest (14). These effects probably play a role in the ability of p53 to act as a tumor suppressor and a number of chemopreventive agents have been shown to exert their anti-tumorigenic activity through p53-dependent mechanisms (1518).

Activation of p53 can result in an increase in its protein half-life and induction of transactivation activity. These events are regulated primarily through post-translation modification. Association with both mdm2 and jun-N-terminal kinase 1 (JNK1) (1921) may affect protein stability by targeting p53 for ubiquitin-dependent degradation. Phosphorylation of serine 15, a substrate site for DNA-dependent protein kinase, disrupted the interaction of p53 with mdm2, which also negatively regulates p53 transcriptional activity (22). Loss of interaction diminished the ability of mdm2 to inhibit p53-dependent transactivation.

A number of different protein kinases can phosphorylate p53 at distinct sites, several of which are associated with changes in transcriptional activity. For example, casein kinase II (CKII) phosphorylates p53 at serine 386 in mouse cells, and replacement of this serine with an alanine led to a loss of growth suppressor activity (23,24). Phosphorylation at serines 9, 15 and 34 in murine p53 may also regulate transactivation since mutation of these sites reduced p53-dependent growth suppression and transactivation (25). However, a study utilizing cells expressing p53 proteins containing mutations at N- and C-terminal phosphorylation sites indicated that phosphorylation at any single site was not essential for transcriptional activity or DNA damage-induced stabilization of p53 (26). N-terminal phosphorylation at multiple sites contributed to stabilization under some conditions, probably by blocking mdm2 binding. Thus, the regulatory role of protein–protein interactions and of phosphorylation at these multiple sites remains to be thoroughly established.

Flavonoids such as apigenin have been reported to induce the expression of p53 protein in mouse fibroblasts (27). To elucidate the cellular response and mechanisms behind cell cycle arrest in keratinocytes, we exposed mouse 308 keratinocytes to apigenin in vitro. Apigenin treatment led to an increase in p53 protein levels due to a prolongation of p53 protein half-life and to induction of transcriptional activity. Thus, apigenin may manifest its chemopreventive actions partially through activation of the p53 pathway.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell culture
The immortalized mouse 308 keratinocyte cell line was derived from Balb/c mouse skin initiated by dimethylbenz[a]anthracene, and contains a point-mutated ras gene, as determined by restriction fragment length polymorphism (28), and a wild-type p53 gene (29). The keratinocytes were cultured in 0.05 mM Ca2+ EMEM medium with 8% chelexed (Bio-Rad Laboratories, Hercules, CA) fetal calf serum. When the cultures reached ~70% confluence, apigenin was added, dissolved in dimethyl sulfoxide (DMSO), to the culture medium to achieve the desired concentration. DMSO levels never exceeded 0.18%. In all experiments the apigenin stock was added to the existing culture medium, rather than to fresh medium, to avoid serum-stimulated effects on cell cycle and protein expression.

Western blot analysis
After treatment, cells were harvested at the indicated times in Tris lysis buffer [TLB; 1 mM Tris, 137 mM NaCl, 25 mM ß-glycerophosphate, 2 mM EDTA, 1 mM Na3VO4, 2 mM sodium pyrophosphate, 1% Triton X-100, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 µg/ml leupeptin, 1 µg/ml aprotinin, 0.2 mM benzamidine, 6.5 mM dithiothreitol (DTT)]. Aliquots (200 µg of cellular protein) were separated by electrophoresis on a 10% SDS—PAGE gel. After transfer to nitrocellulose, the blot was probed with FL393, a rabbit polyclonal anti-p53 antibody or sc-397, a rabbit polyclonal anti-p21 antibody. The secondary antibody was anti-rabbit IgG peroxidase conjugate (all antibodies were from Santa Cruz Biotechnology, Santa Cruz, CA). Western blot analysis was achieved using enhanced chemiluminescence (Amersham, UK). p53 protein half-life studies were performed as described by Chernov et al. (30). Briefly, cells were treated with apigenin for 4 h. Cycloheximide (2 µg/ml) was then added to inhibit further protein synthesis. Cells were harvested in TLB at 15 and 30 min, and 1, 2 and 4 h after cycloheximide addition. Aliquots (300 µg of total cellular protein) were analyzed as described.

Northern blot analysis
Total RNA was isolated using Trizol reagent and method (Gibco BRL, Grand Island, NY). Aliquots (20 µg of total cellular RNA) were loaded onto a 1% formaldehyde agarose gel containing 2.2 mM formaldehyde, 20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA. Formaldehyde agarose gels were transferred to Nytran membrane (Midwest Scientific Inc., St Louis, MO) and the RNA fixed to the membrane by exposure to 120 mJ/cm2 UV in a Stratalinker. Blots were prehybridized at 42°C in 5x SSC, 5x Denhardt's solution, 50% formamide, 1% SDS and 100 µg/ml salmon sperm DNA. Hybridization was carried out at 42°C overnight in prehybridization solution containing 10% dextran sulfate and 106 c.p.m./ml of one of the following randomly primed labeled cDNA probes: 0.93 kb murine p53 cDNA (29) or 1.1 kb human G3PDH (Clontech, Palo Alto, CA). Blots were then washed and subjected to autoradiography. An ~2.5 kb transcript was identified as the p53 mRNA.

Electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared as described (31) and dialyzed against 10.6 mM HEPES, 20% glycerol, 72 mM KCl, 0.8 mM MgCl2, 0.1 M EDTA, 0.5 mM DTT and 0.5 mM PMSF. Oligonucleotides representing the consensus p53-binding site in the p21/waf1 gene promoter (GAACATGTCCCAACACATG TTG) (32) were annealed into double-stranded form and labeled with [{alpha}-32P]dCTP. To determine binding specificity, competition assays were performed using 20x excess cold p21/waf1 oligo and mutant p21/waf1 oligo (GGACATG- CCCGGGCTTTTC) (33). Binding reactions contained nuclear extract (40 µg protein), 1 ng labeled probe and 1.5 µg poly(dI-dC). Binding assays were incubated on ice for 30 min and analyzed on a 4% non-denaturing polyacrylamide gel. The gel was run in 1x Tris–borate–EDTA, dried and analyzed by autoradiography.

Plasmids and reporter gene assay
The luciferase reporter plasmid under control of the mouse p21/waf1 promoter was provided by Xiao et al. (34). This plasmid contained a 4.6 kb fragment (–4542/+113) of the mouse p21/waf1 promoter cloned upstream of luciferase in a pGL3 vector (WT). A double deletion mutant (MUT) was provided by Xiao et al. (34) in which both p53 consensus sequence binding sites were deleted, rendering the promoter inactive to p53-dependent activation. Triplicate wells of 12-well plates were plated at a density of 1.1x105 cells/well and transfected with 400 ng of plasmid reporter construct DNA using the lipofectamine reagent and manufacturer's method (Gibco BRL). Two days after transfection, cells were treated with 70 µM apigenin and assayed by luminometer.

Statistical analysis
Western blotting and EMSA results were analyzed for statistical significance by comparing relative protein levels of cells treated at specific time points to 0 h treated controls using ANOVA. Luciferase assay results were analyzed for statistical significance by comparing WT-transfected cells treated with DMSO with those treated with apigenin for the same time period by ANOVA. In all cases, the analysis corrected for multiple comparisons within experiments.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Apigenin elevated wild-type p53 and p21/waf1 protein in the 308 keratinocyte cell line
Our laboratory is investigating the molecular mechanisms of chemopreventive action of the bioflavonoid apigenin. In this report, we employed western blot analysis to study the effect of apigenin treatment on the p53 tumor suppressor protein. Cultures of the mouse keratinocyte 308 cell line were treated with a range of apigenin concentrations (0–70 µM) and cells were harvested after 8 h. p53 protein levels were assessed using western blot analysis of cellular extracts (aliquots of 200 µg protein). Apigenin produced a dose-dependent increase in p53 protein levels after 8 h of treatment with 70 µM apigenin producing the highest fold induction of the concentrations tested (Figure 1AGo). Concentrations of apigenin >70 µM were not tested because of cytotoxicity. We next analyzed the time course of p53 protein induction in cells treated with 70 µM apigenin. The 70 µM concentration was chosen because previous studies by our laboratory demonstrated that this concentration induced a substantial G2/M arrest in these cells (10) and produced the highest fold induction of p53 protein, as seen in Figure 1AGo. Furthermore, addition of this level of apigenin to culture medium has been shown to result in intracellular levels of apigenin which are comparable with levels achieved in vivo in previous studies (35) while exhibiting negligible toxicity. p53 protein levels in apigenin-treated cells increased in a time-dependent manner over control (DMSO)-treated cells (Figure 1BGo). The cellular level of p53 protein increased significantly after 2, 4, 8 and 10 h of exposure to apigenin by 17-, 27-, 20- and 15.5-fold, respectively, relative to DMSO-treated controls (P < 0.001) as shown in Figure 1CGo. Cellular p53 levels returned to control levels after 24 h of apigenin treatment.



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Fig. 1. Apigenin treatment resulted in a dose- and time-dependent elevation in p53 protein levels in 308 keratinocytes. 308 cells were seeded into 100 mm dishes and cultured until 70% confluent. The cells were treated with 70 µM apigenin, the dishes were harvested at the indicated times, and cell extracts were prepared. Aliquots of cell extracts (200 µg protein) were subjected to gel electrophoresis and western blot analysis of p53 protein was carried out as described in Materials and methods. (A) Western blot analysis showing dose-dependent induction of p53 protein in 308 keratinocytes after 8 h of incubation with the indicated dose of apigenin. (B) Western blot showing time-dependent p53 protein induction by 70 µM apigenin. (C) Quantitative analysis of the time course of p53 protein induction. Autoradiograms were scanned by densitometry and normalized to their DMSO-treated time controls. The graph represents the results of four independent experiments (*P < 0.001).

 
Protein levels of p21/waf1, a downstream target of p53, were also examined. Aliquots of cellular extracts (200 µg protein) were separated by gel electrophoresis and analyzed by western blot. In four separate experiments, treatment of cells with 70 µM apigenin resulted in only a modest time-dependent increase in p21/waf1 protein levels beginning at 4 h and reaching 1.7-fold by 24 h (Figure 2Go). Although the increase in p21/waf1 protein was not statistically significant, the moderate increase in protein levels was consistent in four independent experiments.



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Fig. 2. Apigenin treatment modestly induced p21/waf1 protein levels. 308 cells were seeded into 100 mm dishes and cultured until 70% confluent. The cells were treated with 70 µM apigenin, the dishes were harvested at the indicated times and cell extracts were prepared. Gel electrophoresis of aliquots of the cell extracts (200 µg protein) and western blot analysis of p21/waf1 protein was carried out as described in Materials and methods. (A) Western blot analysis showing induction of p21/waf1 protein in 308 keratinocytes treated with 70 µM apigenin. (B) Quantitative analysis of the time course of p21/waf1 protein induction. Autoradiograms were scanned by densitometry and normalized to their DMSO-treated time controls. The graph represents the results of four independent experiments.

 
Increases in p53 protein are not due to enhanced transcription of p53 mRNA
To investigate the mechanism by which apigenin treatment increased p53 protein levels, we first measured the effect of apigenin on steady state p53 mRNA levels. Apigenin was added to the existing culture medium to avoid adding fresh serum-containing medium that could stimulate serum-related effects on mRNA transcription. Total RNA was isolated from 308 keratinocytes after treatment with 70 µM apigenin for 2, 4, 8, 10 and 24 h. The level of p53 mRNA transcript was analyzed by northern blot analysis. The steady state levels of p53 mRNA remained constant or even significantly decreased after 8 and 10 h of treatment (P < 0.01), then began to return to control levels by 24 h (Figure 3Go).



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Fig. 3. Northern blot analysis of steady state p53 mRNA levels. 308 cells were seeded into 100 mm dishes and cultured until 70% confluent. The cells were treated with 70 µM apigenin and total RNA was harvested at the indicated times. Northern analysis of mRNA was carried out as described in Materials and methods. (A) Northern blot analysis of p53 and G3PDH mRNA in 308 keratinocytes after treatment with 70 µM apigenin for the indicated times. (B) Quantitative analysis of p53 mRNA levels over the time course. Autoradiograms were scanned by densitometry and normalized to G3PDH mRNA levels. The graph represents the results of three independent experiments (*P < 0.01).

 
p53 protein stability markedly increased after apigenin treatment
Since changes in p53 protein levels in cells have been attributed to increases in protein stability (29,30), we investigated the effect of apigenin on p53 half-life. Cultures of 308 keratinocytes were treated with medium containing 70 µM apigenin or 0.18% DMSO (solvent control) for 4 h then 2 µg/ml cycloheximide was added to inhibit new protein synthesis. Extracts were harvested at the indicated times and protein half-life determined. Vehicle-treated cells exhibited a p53 half-life of ~16 min while the half-life of apigenin-treated cells was increased to ~2.2 h (Figure 4Go). Increases in protein half-life were specific to p53 and not a generalized protein phenomenon since apigenin treatment had no effect on p21/waf1 protein half-life despite increased protein levels (data not shown).



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Fig. 4. Apigenin treatment led to an increase in p53 protein half-life. 308 cells were seeded into 100 mm dishes and cultured until 70% confluent. The cells were then treated for 4 h with 70 µM apigenin. Cycloheximide was added and cell extracts were harvested at the indicated times. Gel electrophoresis of aliquots (300 µg protein) of whole cell extracts and western analysis of p53 was carried out as described in Materials and methods. (A) Western blot analysis of p53 protein levels in DMSO- and apigenin-treated cells. (B) Quantitative analysis of p53 protein. Autoradiograms were scanned by densitometry and normalized to p53 levels at time zero. The graph represents the results of three independent experiments.

 
Apigenin treatment increased p53 DNA binding activity
Since p53 can modulate cellular responses by activating transcription of downstream effector genes, we studied the ability of p53 to bind an oligonucleotide containing a consensus DNA binding element for wild-type p53 protein. Cultures of 308 cells treated with 70 µM apigenin for 2, 4, 8, 16 and 24 h were harvested and the nuclear extracts were prepared. Aliquots of the nuclear extracts (40 µg protein) were analyzed by EMSA. p53-specific binding to DNA was moderately enhanced by 1.7-fold after 2 h and maintained through 16 h of treatment (Figure 5AGo). After 24 h of treatment, DNA binding was increased 3.7-fold compared with untreated controls, indicating that apigenin treatment induced transcriptionally active p53 protein. This increase in DNA binding activity was found to be statistically significant (P < 0.05). Oligonucleotide binding specificity was demonstrated by competition assays using 20x excess cold p21/waf1 oligonucleotide and a mutant p21/waf1 variant (Figure 5BGo).



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Fig. 5. Apigenin treatment induced time-dependent increases in p53-specific DNA binding. For (A) and (B), 308 cells were seeded into 100 mm dishes and cultured until 70% confluent. The cells were treated with 70 µM apigenin, the dishes were harvested at the indicated times and the nuclear extracts were isolated. Binding assay analysis of aliquots (40 µg protein) of nuclear extracts was carried out as described in Materials and methods. (A) Increase in p53-specific DNA binding in 308 keratinocyte nuclear extracts after treatment with 70 µM apigenin (*P < 0.05). p53-specific band is indicated with an arrow. (B) Specificity was demonstrated by DNA binding competition assay using 20x excess specific oligonucleotide or non-specific mutant p21/waf1 oligonucleotide.

 
p53 transcriptional activity increased with apigenin treatment
Since DNA binding of p53 can be found in the absence of transactivation (36), we investigated the transcriptional activity of accumulated p53 after apigenin treatment. Triplicate cultures of 308 cells were transfected with a p53-dependent luciferase reporter construct (WT) or a double deletion mutant construct in which both p53-binding sites had been deleted (MUT). Cells were treated 48 h after transfection with 70 µM apigenin for 4, 8 and 24 h. Apigenin treatment of WT reporter construct-transfected cells resulted in the stimulation of transactivational activity in a time-dependent manner. Transactivational activity significantly increased ~2.5-fold after 24 h exposure (Figure 6Go) in two independent experiments (P < 0.05). This time course was in agreement with the DNA binding studies. DMSO treatment of WT transfected cells produced no induction of transactivational activity. Cells transfected with MUT luciferase constructs that lack p53 binding sites did not show any increase in transactivation after exposure to DMSO or apigenin, confirming that apigenin-induced stimulation of transactivation was p53-dependent.



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Fig. 6. Apigenin treatment stimulated p53-dependent transcriptional activity. 308 keratinocytes were seeded in 12-well plates and transfected with a luciferase construct containing the p21/waf promoter region containing p53 binding sites (WT) or a double deletion mutant (MUT) in which both p53 binding sites had been removed. Two days after transfection, cells were treated with 70 µM apigenin and harvested at the indicated times. Luciferase activity was measured and the results expressed as relative p53-dependent luciferase activity. Luciferase assays were performed with triplicate dishes and the graph is representative of two independent experiments (*P < 0.05).

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Previous studies demonstrated that topical application of apigenin, a non-mutagenic bioflavonoid, inhibited UV-induced tumors in mice in vivo (6) and addition of apigenin to culture medium caused both a G1 and a G2/M arrest in vitro (8,10). In this paper, we explored possible mechanisms of anti-tumorigenicity by investigating the activation of the cell cycle regulatory proteins p53 and p21/waf1. Apigenin treatment led to cellular increases of both proteins in a time-dependent manner. We also show that apigenin induced the stabilization and transcriptional activity of the tumor suppressor protein p53. p53 protein plays a major role in the activity of a variety of chemopreventive agents including other polyphenolic compounds, isothiocyanates, retinoid derivatives and carotenoids (1518), underscoring its importance in tumor prevention. Our results indicate that p53-initiated events may play a role in cell cycle arrest induced by apigenin and subsequent inhibition of cell proliferation and tumorigenesis.

Our experiments showed that apigenin treatment led to a dose- and time-dependent increase in p53 protein in 308 keratinocytes. This increase in p53 protein was not reflected by an increase in mRNA transcription. Steady state levels of p53 mRNA levels remained constant or even decreased, suggesting that apigenin stimulated an increase in p53 protein levels independent of the accumulation of p53 mRNA. A similar finding was reported by Mosner et al. (37), who observed that in growth-arrested mouse fibroblasts re-entering the cell cycle, p53 protein levels increased at a time when mRNA levels were declining. Furthermore, p53 protein levels accumulated even in the presence of transcriptional inhibitors (37), supporting our observation that apigenin-induced accumulation of p53 protein could occur in the absence of increased mRNA levels. It has also been demonstrated in normal mouse fibroblasts that p53 mRNA levels decrease during G0 and G1 arrest (38). It is unclear in our system if ensuing G2/M arrest triggered the down-regulation of p53 mRNA. G2/M arrest commenced after 8 h of apigenin treatment and was pronounced by 24 h (data not shown). p53 mRNA levels initially decreased coincident with increasing growth arrest; however, mRNA levels recovered even as a greater population of cells underwent G2/M arrest. This suggests that cell cycle arrest and changes in p53 mRNA were not causally related. We found that apigenin-induced elevation of p53 protein levels was due to an 8-fold increase in p53 protein stability. The mechanism of p53 protein stabilization is currently being investigated and could involve phosphorylation of discrete residues (26,39), altered protein interactions (19,20) or inhibition of p53 ubiquitination as has been suggested by studies from other laboratories (21,40).

Our studies further ascertained that p53 protein was transactivationally activated by apigenin. We utilized two different approaches to demonstrate an effect on p53 transactivation. Gel mobility shift assays were used to show increased DNA binding activity in nuclear extracts from apigenin-treated cultures. In addition, transient transfection studies clearly confirmed transactivational activation of the p21/waf1 promoter in apigenin-treated cells, indicating that apigenin treatment induced functionally active p53 protein. Interestingly, we observed that increases in cellular p53 protein levels did not correspond temporally with increases in p53 transactivational activity. However, an increase in the amount of p53 protein is not always accompanied by increased transactivation of genes (41). Hupp et al. (41) demonstrated that latent p53 can be activated by post-translational modification of the C-terminal negative regulatory domain by the pAb421 antibody in the absence of increased protein levels. Incongruity between the transcriptional activity of p53 and its protein levels has also been reported in UV-irradiated fibroblasts (42). p53 protein level and protein half-life have even been reported to decrease in association with increased transcriptional activity in differentiating primary mouse epidermal keratinocytes (43). Additionally, hyperphosphorylation of p53 induced by okadaic acid treatment increased cellular levels of p53 in association with enhanced DNA binding but resulted in decreased transcriptional activity (36). In our model, both DNA binding and transactivation of p21/waf1 promoter were induced maximally after stabilized p53 levels had returned to control levels.

Activation and stabilization of p53 appear to be independently regulated by different phosphorylation events (30) and protein interactions. PKC, which has been shown to phosphorylate p53 (44), can activate DNA binding by recombinant p53 (45). Furthermore, activation of PKC led to increased phosphorylation of wild-type p53 accompanied by increased DNA binding and cell cycle arrest (46). Mutation of the CKII phosphorylation site, serine 386, abrogated the anti-proliferative activity of p53 (24), suggesting a role of CKII in regulation of p53 activity. Phosphorylation at serine residues 6, 15 and 34 of mouse p53 has been shown to enhance transactivation and growth suppression mediated by p53 (25), while mutation of serine 15 to an alanine resulted in an increased p53 half-life but lower steady state levels of p53 (47). This indicates that phosphorylation of serine residues in the N-terminus of p53 may influence both transactivation and stability. It is possible that apigenin treatment may activate various protein kinases or phosphatase which regulate p53 at different times, leading to the observed disparity in time course induction of protein levels and transcriptional activity.

Previous results from our laboratory demonstrated that apigenin induced G2/M arrest in 308 keratinocytes (10). We have also shown that this arrest is partially the result of apigenin-induced inhibition of cyclin B1-associated cdc2p34 activity (10). Results presented in this report support the hypothesis that apigenin-induced cell cycle arrest may be partially mediated by p53 and p21/waf1. p53 has been shown to play an important role in G1 arrest and an emerging role in G2/M arrest (11) when inducibly expressed. In the absence of DNA damage, induction of p53 led to both a reversible G1 and a G2/M arrest in tetracycline-responsive human fibroblast cell lines (12), supporting a role for p53 in cell cycle arrest. Synchronization of these same cells in S-phase led to a predominant G2/M arrest. Thus, p53 induced cell cycle arrest may be dependent upon the phase of the cell cycle in which the cells reside when p53 is activated. p21/waf1, traditionally thought to regulate G1 arrest via the inhibition of Cdks associated with cyclin D1 and E, may also control the G2/M phase transition (48). Dulic et al. (48) demonstrated that p21/waf1 transiently accumulated in the nucleus as cells neared the G2/M boundary and associated with both cyclin A–Cdk and cyclin B1–Cdk complexes, promoting a pause late in G2. Thus, p53 and p21/waf1 may partially mediate the apigenin-induced G2/M cell cycle arrest observed in the 308 keratinocytes and the G1 arrest induced in other cell types.

Activation of cell cycle arrest, thereby mimicking the cell's protective DNA damage response, could be one chemopreventive consequence of apigenin treatment. While this arrest may involve p53 and p21/waf1, these molecules may also induce other, as yet uncharacterized, effects that contribute to the chemopreventive effect of apigenin. p53 has been shown to play a role in apoptosis and DNA repair. Although we did not observe any apoptosis in 308 cells as measured by DNA laddering, TUNEL, flow cytometry and morphological changes even after 3 days of exposure (data not shown), exposure to higher concentrations of apigenin have led to apoptotic death in mouse C3H10T1/CL8 fibroblasts (27). The effect of apigenin on DNA repair has not been investigated; however, preactivation of p53 may enhance DNA repair capacity and cell survival in response to subsequent DNA damage. This effect has been demonstrated after exposure of human skin cells to thymidine dinucleotides which mimic DNA damage, activate p53 and upregulate genes involved in DNA repair resulting in improved DNA repair and cell survival following UV irradiation (49).

In this report we have further elucidated the mechanisms underlying apigenin-induced chemoprevention. p53-mediated events may contribute to inhibition of tumor growth after apigenin treatment. Further investigation of the mechanism by which p53 is stabilized could delineate the effect of apigenin on protein kinases and phosphatases. If diverse targets exist, apigenin could exert chemopreventive effects through multiple pathways. This multiplicity could be exploited in treating both wild-type and mutant p53-containing tumors. In light of the rising number of patients diagnosed with skin cancer and the fact that individuals diagnosed with basal or squamous cell carcinomas of the skin are at an increased risk for developing a second skin cancer, additional studies to identify apigenin's underlying mechanism of chemopreventive action are merited.


    Acknowledgments
 
This research was supported by NIH grant CA72987.


    Notes
 
4 To whom correspondence should be addressed Email: jpelling{at}kumc.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 20, 1999; revised December 2, 1999; accepted December 20, 1999.