Protein Phosphatase-2 A Is a Target of Epigallocatechin-3-Gallate and Modulates p 53-Bak Apoptotic Pathway

(-)-Epigallocatechin-3-gallate (EGCG) is a well-known chemoprevention factor. Recent studies have revealed that EGCG triggers cancer cells undergoing apoptosis through p53dependent pathway. How EGCG activates p53-dependent apoptosis is not fully understood. In the present study using JB6 cell as a model system, we have shown that EGCG can negatively regulate protein serine/threonine phosphatase-2A (PP-2A) to positively regulate p53-dependent apoptosis. First, EGCG at physiologic levels down-regulates PP-2A at the protein and enzyme activity levels. Second, EGCG induces apoptosis of JB6 cells, which is associated with hyperphosphorylation of p53 and up-regulation of the proapoptotic gene, Bak . DNA sequence analysis, gel mobility shifting, chromatin immunoprecipitation, and reporter gene activity assays revealed that p53 directly controls Bak in JB6 cells. Knockdown of p53 and Bak expression with RNAi substantially inhibits EGCG-induced apoptosis. Third, PP-2A directly interacts with p53 and dephosphorylates p53 at Ser-15 in vitro and in vivo. Fourth, overexpression of the catalytic subunit for PP-2A down-regulates p53 phosphorylation at Ser15, attenuates expression of the downstream proapoptotic gene, Bak , and antagonizes EGCG-induced apoptosis. Inhibition of PP-2A activity enhances p53 phosphorylation at Ser-15 and up-regulates Bak expression to promote EGCG-induced apoptosis. Finally, in the p53 / H1299 and p53 H1080 cells, EGCG down-regulates PP-2A similarly but induces differential apoptosis. In summary, our results show that (a) PP-2A directly dephosphorylates p53 at Ser-15; (b) P53 directly controls Bak expression; and (c) EGCG negatively regulates PP-2A. Together, our results show that EGCGmediated negative regulation of PP-2A is an important molecular event for the activation of p53-dependent apoptosis during its chemoprevention. [Cancer Res 2008;68(11):4150–62]

PP-2A exists in cells in two major forms: holoenzyme and core enzyme (16,22).The core enzyme of PP-2A comprises a 65-kDa scaffolding A subunit and a 36-kDa catalytic C subunit (22).PP-2A activity is largely regulated by the binding of one of at least 18 regulatory B subunits to the A to C core enzyme (16,22).Previous studies have revealed that both the scaffold A subunit and the regulatory B subunit are involved in regulation of carcinogenesis (19,23).The evidence for the involvement of scaffold A subunits in tumorigenesis is derived from the discovery that both Aa and Ah subunits are found mutated or deleted in 15% lung, breast, colon, and skin cancers (24,25).The mutation or deletion of either of these subunits likely abolishes its ability to bind the regulatory B unit, or both B and C subunits (26,27).On the other hand, the implication of the regulatory B subunits in tumorigenesis is reasoned from the observations that Ras-mediated cell transformation does not occur in the absence of either Ras or small T antigen in both human fibroblasts or embryonic kidney cells (28,29).It was observed that the function of the small T antigen is to displace the regulatory B subunit(s) and, thus, to suppress PP-2A activity (30,31).However, whether the catalytic subunit alone can directly regulate carcinogenesis remains to be further explored.In the present study, we present first evidence to shown that the catalytic subunit of PP-2A (PP-2Acs) alone plays an important in modulating carcinogenesis through dephosphorylation of p53 at Ser-15 and inhibition of p53-Bak pathway.
In addition, PP-2A is an important target in EGCG-mediated chemoprevention.
Generation of stable transfectants.JB6 Cl41 cells were transfected with various mock and expression constructs including pCI-neo vector, the PP-2Acs expression construct, the small hairpin control construct for p53 RNAi (shmock), and the small hairpin p53 silence construct (shp53) using Lipofectamine 2000 following the manufacturer's instructions.The stable transfectants were obtained through G418 selection (400 Ag/mL) or puromycin selection (2 Ag/mL) and further confirmed by reverse transcription-PCR (RT-PCR) and Western blot analysis as described before (32).The selected clones of both vector and PP-2A-transfected JB6 Cl41 were cultured in the same conditions described above plus 400 Ag/mL G418.The selected clones of both shmock and shp53-transfected JB6 C141 cells were cultured in the same medium conditions except that the 400 Ag/mL G418 was replaced with 2 Ag/mL puromycin.
Cell treatment.JB6 Cl41, H1299, and H1080 cells or various stable lines of transfected JB6 cells were seeded onto 100-mm dish, and the cells were serum starved for 24 h in 0.1% FBS MEM before EGCG treatment.After starvation, the cells were treated with different concentrations of EGCG (0-80 Amol/L) in the absence (Supplementary Fig. S1; Fig. 1A) or presence of 20 ng/mL 12-O-tetradecanoylphorbol-13-acetate (TPA).After 24 h treatment with EGCG and TPA, the cells were harvested for flow cytometry analysis (32,33), Western blot analysis (32,33), and/or assay of phosphatase activities (33).To inhibit protein serine/threonine phosphatase activities, JB6 C141 cells were pretreated with 0.01% DMSO or 10 to 200 nmol/L okadaic acid, followed by different concentrations of EGCG (0, 10, and 20 Amol/L), and 20 ng/mL TPA for an additional 12 h.After treatment, the cells were harvested for cell flow cytometry and/or Western blot analysis.The pCI-neo, pCI-PP-2Acs, shmock, and shp53 transfectants were also seeded onto 100-mm dish.After serum starvation for 24 h in 0.1% FBS and Figure 1.Regulation of PP-2Acs by EGCG at the mRNA, protein enzyme activity, and cellular localization levels.Aa and Ba, EGCG down-regulates expression of the mRNA and protein for the catalytic subunit of PP-2A.Mouse JB6 Cl41 cells were treated with 0, 10, 20, and 80 Amol/L EGCG in the absence (A) or presence (B) of 20 ng/mL TPA for 24 h with 0.1% serum in culture medium, then harvested for extraction of total RNA and proteins.RT-PCR and Western blot analysis were conducted as described in the materials and methods.Bb and Bb, EGCG down-regulates the enzyme activity of PP-2A.JB6 C141 cells were treated as in Aa or Ba and then harvested for extraction of total proteins.PP-2A activity was assayed as previously described (33,34).Note that the protein and enzyme activity were clearly down-regulated at 10 to 20 Amol/L of EGCG (A and B).Ca to Cd, immunocytochemistry analysis of PP-2Acs localization in JB6 C141 cells.Mouse JB6 Cl41 cells were treated with 0 Amol/L (Ca ), 10 Amol/L (Cb ), 20 Amol/L (Cc), and 80 Amol/L (Cd ) EGCG for 24 h with 0.1% serum in culture medium, then proceeded for immunocytochemistry as described in the Materials and Methods.Note that EGCG at the 10 to 20 Amol/L promotes translocation of PP-2Acs from cytoplasm to nucleus (Cb and Cc).However, under treatment of 80 Amol/L EGCG, PP-2Acs were relocated to cytoplasm around nucleus (Cd ).D, fractionation-linked Western blot analysis of the PP-2Acs in the cytoplasm (C ) and nucleus (N ) of JB6 cells without (a) and with (b ) 20 Amol/L EGCG treatment for 24 h.Isolation and analysis of nuclear and cytoplasmic proteins were conducted as previously described (35).Note that EGCG treatment promotes nuclear localization of PP-2Acs.Shown here are typical results of three independent experiments.Figure 2. EGCG induces p53-and Bak -dependent apoptosis of JB6 cells.JB6 Cl41 cells were treated in the same way as described in Fig. 1B , and then harvested for analysis of apoptosis by flow cytometry (Aa-Ac).Ba, differential dose-dependent response to EGCG by the p53 (+/+) JB6 cells and p53 (+/+) H1080 cells, as well as the p53 (À/À) H1299 cells in the presence of 20 nmol/L TPA.The three types of cells were cultured to the same density then subjected to serum starvation and EGCG treatment as described in Fig. 1B.Note that EGCG-induced apoptosis is substantially attenuated in the p53 (À/À) H1299 cells.Bb, Western blot analysis of p53 and its downstream gene products, p21, Bax, Bak .JB6 C141 cells were treated in the same way as described in Fig. 1B and then harvested for extraction of total proteins, which were used for Western blot analysis as previously described (32)(33)(34).Note that EGCG at physiologic levels up-regulates expression of p53, p21, and Bak proteins and also induces p53 hyperphosphorylation at Ser-15.Shown here are typical results of three independent experiments.Bc, RT-PCR analysis of the p53 downstream genes.RT-PCR procedures were described in the Materials and Methods.The PCR results were averaged after scanning as described before (35).C, silence of p53 substantially blocks EGCG-induced apoptosis.Ca, Western blot analysis to determine the p53 level in JB6 C141 cells harboring either the control vector (shmock) or the p53 shRNA expression construct (shp53 ).The p53 RNA silenced by hairpin RNA oligo against p53 was described in the Materials and Methods.Western blot analysis was conducted as previously described (32)(33)(34).Cb, quantitative comparison of EGCG-induced apoptosis in the presence of p53 (shmock ) or knockdown of p53 (shp53 ) through cell flow cytometry analysis.D, knockdown of Bak substantially attenuates EGCG-induced apoptosis.Da, Western blot analysis to determine the Bak level in JB6 C141 cells transfected with mock siRNA or Bak siRNA.The Bak RNA silenced by siRNA for Bak was described in the Materials and Methods.Western blot analysis was conducted as previously described (32)(33)(34).Db and Dc, comparison of EGCG-induced apoptosis in the presence of Bak (mock siRNA ) or knockdown of Bak (Bak siRNA ) through cell flow cytometry analysis.Note that Bak knockdown caused a 45% attenuation of the EGCG-induced apoptosis.Shown here are typical results of three independent experiments.

Cancer Research
Cancer Res 2008; 68: (11).June 1, 2008 400 ug/mL G418 MEM or 2 Ag/mL puromycin, the cells were treated with different concentrations of EGCG (0-40 Amol/L), in the presence of 20 ng/mL TPA before harvesting for cell flow cytometry analysis and/or Western blot analysis.As a control for EGCG treatment, JB6 cells were also treated with 0 to 500 nmol/L Adriamycin for 24 h in the presence of 0.1% FBS MEM.
Western blot analysis.Western blot analysis was conducted as previously described (32)(33)(34).Total proteins were extracted from parent JB6, H1299, and H1080 cells, or transfected JB6 cells with or without treatment by okadaic acid, EGCG, and/or TPA, or parent JB6 cells with or without treatment by Adriamycin.Cytoplasmic and nuclear proteins were isolated as previously described (34) except that the nuclear soluble and insoluble portions are combined.The protein concentration was determined as described before (32,33).Fifty or 100 Ag of total proteins, cytoplasmic, or nuclear proteins were used for each lane of loaded sample.The protein blots were blocked with 5% milk in TBS buffer (32,33) overnight at 4jC and then incubated for 1 h or overnight with antibodies against Bax, p21, total p53 (Santa Cruz Biotechnology, Inc.), puma, noxa, and phospho-p53-Ser-15 and phospho-p53-Ser-20 (Cell Signaling Technology), Bak and PP-2Acs (Upstate, Inc.), and h-actin antibody (Sigma) at a dilution of 1:200 to 3,000.The secondary antibody was anti-mouse IgG, anti-rabbit IgG, or anti-goat IgG (Amersham Biosciences) at a dilution of 1:1,000 to 3,000.Immunoreactivity was detected as described before (32)(33)(34).
Flow cytometry analysis.The apoptosis rate of parent JB6, H1299, and H1080 cells, or transfected JB6 cells after various treatment was determined using an Annexin V-FITC Apoptosis Detection kit as previously described (32,33).Briefly, the treated cells were trypsinized, washed twice with icecold Dulbecco's PBS, and incubated at room temperature for 15 min in the dark with Annexin V-conjugated FITC and propidium iodide in the binding buffer provided.Stained cells were analyzed by flow cytometry using the FACSCalibur (BD Biosciences) as previously described (32,33).
Assays of protein serine/threonine phosphatase activities.The protein phosphatase activities in the parent and transfected JB6 cells after different treatment were assayed as previously described (33) using a kit from New England Biolabs, Inc.
Immunocytochemistry analysis.JB6 C141 cells were cultured in slide chamber (Fisher Scientific) at the same condition as described above.After Establishment of the stable expression clones, pCI-neo-JB6 and pCI-PP2Acs-JB6, was described in the Materials and Methods.B, cell flow cytometry of both vector-(Ba) and pCI-PP-2Acs (Bb)-transfected clones were treated with 20 Amol/L EGCG, as described in Fig. 1B , for 24 h and then harvested for cell flow cytometry as previously described (32,33).C, quantitative comparison of EGCG-induced apoptosis in the vector-transfected cells (vector ) or PP-2Acs-transfected cells (PP-2Acs ).Shown here are typical results of three independent experiments.D, overexpression of PP-2A blocks EGCG-induced up-regulation of p53, p21, and Bak proteins, and p53 phosphorylation at Ser-15.Both pCI-neo-JB6 and pCI-PP-2Acs-JB6 cells were treated as described in Fig. 1B and then harvested for Wastern blot analysis as previously described (32)(33)(34).Note that overexpression of PP-2Acs attenuates EGCG-induced up-regulation of p53, p21, and Bak protein levels, and p53 phosphorylation at Ser-15.Shown here are typical results of three independent experiments.
95% confluence, the cells were fixed in 1% formalin in PBS for 10 min, then rinsed with PBS thrice, incubated with 10% normal blocking rabbit serum in PBS for 20 min to suppress nonspecific binding of IgG.The blocked slides were further incubated with anti-PP-2Acs for 60 min (1:200), followed by washing thrice with PBS, and then incubated with Texas-red-linked anti-rabbit IgG (1:250) for 45 min.After three washes in PBS, the slides were mounted with 90% glycerol in PBS and photographed under Leica Fluorescence Microscopy.
In vitro phosphorylation of the p53 fusion proteins.The fusion proteins of glutathione S-transferase (GST)-p53 and GST-p53-S15A were prepared and phosphorylated by DNA-dependent protein kinase (DNA-PK) as previously described (33).Equal amounts of GST-p53 and GST-p53-S15A were each incubated with DNA-PK in the presence of g-32 P-ATP for 30 min at 30jC.The reaction was stopped by adding 1/5 volume of 100% trichloroacetic acid (TCA) to precipitate the reaction mixture, which was then dissolved into 40 AL 1 Â protein phosphatase assay buffer for dephosphorylation assay (33).
In vitro dephosphorylation assays.The in vitro dephosphorylation assays were conducted as previously described (33,34).
In vivo dephosphorylation assays.The in vivo dephosphorylation assays were conducted as described before (33,34).Briefly, JB6 cells were labeled with [ 32 P]-P i (200 ACi/mL) in phosphatase-free DMEM and subjected to 80 Amol/L EGCG treatment for 4 h in the presence of 20 ng/mL TPA.Then, total proteins were extracted for immunoprecipitation with an antibody against phospho-p53-Ser-15.The immunoprecipitated protein complex was mixed with phosphatase reaction buffer and incubated at 30jC for 30 min.After TCA precipitation, the supernatant fraction was recovered for counting the release of free 32 P in a scintillation counter.Immunoprecipitated sample with anti-h-actin antibody was also used for the parallel dephosphorylation assay (mock).The dephosphorylation assay was conducted in the absence or presence of PP-2A-specific inhibitor, PP2A-I1; or PP-1-specific inhibitor, PP1-I2; or PP-2B-specific inhibitor, CSA, or okadaic acid to block both PP-1 and PP-2A.
Silence of p53, Bak, and PP-2Acs.For stable knockdown of p53 expression in JB6 cells, the pSHAG-MAGIC vector expressing shRNA against p53 expression and the nonsilencing shRNA pSHAG-MAGIC vector as negative control were obtained from the Open Biosystem.Transfection of JB6 cells was performed using a Lipofectamine 2000 agent as described previously (33,34).Stably transfected cell clones were selected with puromycin (2 Ag/mL).For knockdown of Bak and PP-2Acs expression, the siRNA oligos were obtained from Santa Cruz Biotechnology and the transfection was conducted as previously described (33,34).
Gel mobility shifting assays.Gel mobility shifting assays were conducted as previously described (35).The oligos used were as follows: 5 ¶-GTTGGCTTGCCTGCCTCTGCCTCT-3 ¶ for mouse Bak gene p53 binding site and 5 ¶-GTTCCACCAAAGAAATCCAAAGCT-3 ¶ for mutated p53 binding site.Forty micrograms of nuclear extracts prepared from JB6 cells treated with 40 Amol/L EGCG and 20 ng/mL TPA for 12 h were incubated with 1 Â 10 5 CPM of 32 P-labeled double-stranded synthetic oligonucleotides for 30 min at 30jC in a binding shifting buffer (35).For competition experiments, 50-fold of the unlabeled wild-type or mutant double-stranded synthetic oligonucleotides were preincubated with the nuclear extracts for 10 min before the labeled probe was added into the reaction.In the precleared experiments, 10 Ag antibody against p53 was preincubated with the nuclear extracts on ice for 10 min before addition of the 32 P-labeled oligonucleotides.After the binding reaction, the mixtures were loaded onto 5% native PAGE and detected by autoradiography.
Analysis of transient gene expression.For reporter gene activity, the constructs of the luciferase reporter gene driven by the Bak gene promoters with a wild-type or mutant p53 binding sites inserted at À74 were each introduced into JB6 cells with an internal control plasmid using Lipofectamine 2000 as described above.Two hours after transfection, the transfected cells were treated with 20 ng/mL TPA alone or 20 Amol/L EGCG in the presence of 20 ng/mL TPA for additional 22 h before harvested for assays of luciferase activity using a kit from Promega as described previously (33,34).For p53 dose-dependent response, the Bak-luc construct containing a wild-type p53 site, and the internal control plasmid plus 0 to 1,000 ng of pCMV-p53 plasmid were cotransfected into both JB6 and H1299 cells, the transfected cells were harvested after 24 h, and the harvested cell extracts were used for assay of luciferase activity as described above.
Chromatin immunoprecipitation assay.JB6 cells were grown to 95% confluence, then starved in 0.1% serum overnight, and followed by treatment with 20 Amol/L EGCG for 24 h in the presence of 20 ng/mL TPA.Approximately 2.0 Â 10 7 cells were incubated with 1% formaldehyde for 10 min at room temperature for crosslinking, which was terminated by washing the cells with 4 mL of 1.25 mol/L glycine solution.The cells were further washed with cold PBS twice and then scraped into 1 mL of chromatin immunoprecipitation (ChIP) sonication buffer [50 mmol/L Tris-HCl (pH 8.1), 1% Triton X-100, 0.1% sodium deoxycholate, 5 mmol/L EDTA, and 150 mmol/L NaCl] containing the protease inhibitor cocktail.These lysates were sonicated 15 times for 10 s each time to generate DNA fragments that ranged in size from 200 to 1,000 bp.The sheared chromatinlysed extracts were incubated with either 5 Ag of anti-p53 antibody or 5 Ag of normal IgG overnight at 4jC, and then incubated for an additional 1 h with 30 AL protein A/G agarose beads.The immunoprecipitates were washed by cold ChIP sonication buffer 2Â and cold PBS 2Â, then suspended in the elution buffer [Tris-EDTA buffer (pH 8.0) and 1% SDS], and incubated overnight at 65jC, and an additional 2 h at 55jC with 100 Ag of protease K to reverse proteins/DNA crosslinks.Finally, these samples were processed for DNA purification by phenol-chloroform extraction and ethanol precipitation.PCRs were performed in 25 AL with 1/1,000 of input DNA or 1/50 of the immunoprecipitates with the following primers: 5 ¶-CTCTGCCTAGCTGCCTCAATG-3 ¶ ( forward) and 5 ¶-GAGACCACCGT-CACTTGTCAC-3 ¶ (reverse), which generates a fragment of 383 bp.As a positive control, a pair of oligos derived from p21 promoter containing the p53 binding site were designed as follows: ( forward) 5 ¶-AGGGTAAAGGCA-CAGGAGGT-3 ¶ and (reverse) 5 ¶-ACAGCTTCTCCAAA-GCAGGA-3 ¶, which produces a fragment of 478 bp.PCR was conducted with following specifications: 94jC 5 min, (94jC 30 s, 52jC 30 s, and 72jC 30 s) Â 32, 72jC 5 min, and the PCR products were separated on an 1.5% agarose gel and stained with ethidium bromide.

Results
PP-2A is regulated by EGCG at multiple levels.To investigate whether EGCG acts on protein serine/threonine phosphatases during its chemopreventive function, we examined the expression, activity, and localization of PP-2A after treatment by different concentrations of EGCG in the absence (Fig. 1Aa) or presence of 20 ng/mL TPA (Fig. 1Ba).As shown in Fig. 1Aa and Ba, although the mRNA for PP-2Acs remained relatively unchanged under A, the SDS-PAGE analysis of the fusion proteins for GST-p53 and GST-p53-S15A before (left ) and after (right ) glutathione column purification.B, in vitro dephosphorylation assays by PP-2A.The two substrates, GST-p53 and GST-p53-S15A, were labeled with DNA-PK and precipitated by TCA after labeling.Then equal amount of each protein (2 Ag protein for each sample) was mixed with 1Â PP-2A dephosphorylation buffer and 10 ng PP-2Acs (obtained from Calbiochem, Inc.) in the absence or presence of specific PP-2A inhibitor, PP2A-I1.The mixture was incubated 10 min at 30jC.After the reaction, the released free 32 P was counted.The final results were averaged from three independent experiments in which the free 32 P released from each dephosphorylation reaction was recorded.C, reciprocal immunoprecipitation (IP ) to show that PP-2Acs and p53 form interacting complex.JB6 cells were harvested for extraction of total proteins, which were used for immunoprecipitation with anti-PP-2Acs antibody (1 and 2 ) or anti-total p53 antibody (3 and 4 ).The immunoprecipitated samples were then subjected to Western blot analysis as described before (32)(33)(34).The supernatant represents the sample after immunoprecipitation.Shown here are typical results of three independent experiments.D, in vivo dephosphorylation of p53 at Ser-15 by PP-2A.JB6 cells were labeled with [ 32 P]-P i (200 ACi/mL) in phosphatase-free DMEM for 30 min and then subject to 80 Amol/L EGCG treatment for another 4 h in the presence of the [ 32 P]-P i (200 ACi/mL).Total proteins were extracted for immunoprecipitation with an antibody against phospho-p53 at Ser-15.The immunoprecipitated protein complexes were mixed with phosphatase reaction buffer in the presence or absence of various inhibitors and then incubated at 30jC for 10 min.After TCA precipitation, the supernatant fraction was recovered for counting the release of free 32 P in a scintillation counter.Immunoprecipitated sample with antiactin antibody was also used for the parallel dephosphorylation assay (mock ).The dephosphorylation assays were conducted in the absence (extract ) or presence of PP-1 inhibitor, PP1-I2 (Extract +PP1-I2 ) or 200 nmol/L okadaic acid (Extract +OA ), PP-2A inhibitor, PP2A-I1 (Extract +PP2A-I1 ), and PP-2B inhibitor, 250 nmol/L cyclosporin A (Extract +CSA ).The results shown here are averaged from three independent experiments.Note that the inhibitor blocking PP-2A but not PP-2B was able to inhibit f75% dephosphorylation.In contrast, the specific inhibitor for PP-1 only inhibited f25% of dephosphorylation, indicating that in JB6 cells, PP-2A is the major phosphatase that dephosphorylates p53 at Ser-15.IB, immunoblot.
treatment of 10 and 20 Amol/L EGCG, significant down-regulation of the PP-2Acs mRNA was observed after the JB6 cells were treated by 40 to 80 Amol/L EGCG (Fig. 1Aa and Ba, 1).As a comparison, EGCG treatment at the experimental concentrations did not affect the level of the mRNA for h-actin (Fig. 1Aa and Ba, 2).At the protein level, however, 20 Amol/L EGCG induced a significant down-regulation of the PP-2Acs protein (Fig. 1Aa and Ba, 3) and also PP-2A activity (Fig. 1Ab and Bb).Western blot analysis revealed that EGCG also down-regulates PP-2A in other cancer cells (Supplementary Fig. S1A).Although 10 Amol/L EGCG only caused slight change in the PP-2Acs protein level and activity (Fig. 1A and B), EGCG at this concentration did cause change in PP-2Acs localization, and majority of the PP-2Acs was concentrated to the nuclei of the treated JB6 cells (Fig. 1Ca and Cb).Under treatment of 80 Amol/L EGCG, however, PP-2Acs was found released from the nuclei of the treated cells and moved into the cytoplasm around the nuclei of the treated cells (Fig. 1Cd).The differential localization induced by physiologic level of EGCG was further confirmed through differential fractionation-linked Western blot analysis (Fig. 1Da and Db).TPA had little effects on EGCG-mediated regulation of PP-2A (Fig. 1A and B).Thus, EGCG at both physiologic (10-20 Amol/L) or nonphysiologic concentration (80 Amol/L) regulates PP-2Acs at different aspects.
EGCG induces p53-dependent apoptosis in JB6 cells.Previous studies have shown that EGCG-induced p53-dependent apoptosis in cancer cells (9).To confirm this function of EGCG in JB6 cells, we first treated JB6 cells with different concentration of EGCG and examined its role in triggering apoptosis of JB6 cells.As shown in Fig. 2Aa to Ac, cell flow cytometry analysis revealed that in the presence of 20 ng/mL TPA, EGCG induced dose-dependent apoptosis of JB6 cells, whereas TPA alone only caused background apoptosis.Analysis of EGCG-induced apoptosis in JB6 cells and two lung cancer cell lines, p53 (+/+) 1080 and p53 (À/À) H1299 cells, revealed that lack of functional p53 distinctly attenuates EGCG-induced apoptosis (Fig. 2Ba).
To explore if EGCG activates p53-dependent apoptosis in JB6 cells, we have examined the expression level and phosphorylation status of the tumor suppressor, p53, under treatment of different concentrations of EGCG.As shown in Fig. 2Bb, under normal culture condition, the p53 level was barely detectable in JB6 cells.Treatment of JB6 cells with EGCG from 10 to 40 Amol/L induced dose-dependent up-regulation of p53 and also dose-dependent hyperphosphorylation at Ser-15.However, phosphorylation of p53 at Ser-20 was not detectable (Supplementary Fig. S2A).In contrast, treatment of JB6 cells by Adriamycin induced hyperphosphorylation of p53 at both Ser-15 and Ser-20 of p53 (Supplementary Fig. S2C).Associated with up-regulation of both total p53 and also hyperphosphorylation of p53 at Ser-15, the CDK inhibitor, p21, and the proapoptotic gene, Bak, were also up-regulated at the protein level (Fig. 2Bb) and mRNA level (Fig. 2Bc) after EGCG treatment.A mutation from serine to alanine at Ser-15 of p53 caused clearly reduced induction of Bak in transfected H1299 cells (Supplementary Fig. S1B).On the other hand, the proapoptotic gene, Bax, was only slightly up-regulated due to its presence of high background level in JB6 cells.Examination of EGCG-induced expression of additional p53 target genes revealed that Puma and Noxa were hardly detectable in both untreated and treated JB6 cells at protein levels (Supplementary Fig. S2A).At the mRNA level, however, although Noxa mRNA was undetectable, the Puma mRNA was present but the level was lower than Bax or Bak mRNAs (Supplementary Fig. S2B).As comparison, treatment of JB6 cells by Adriamycin induced up-regulation of the proteins and mRNAs for p21, Bak, Bax, and Puma (Supplementary Fig. S2C and D, respectively).Again, Noxa mRNA and protein were still undetectable (Supplementary Fig. S2C and D).Together, these results suggest that EGCG induces apoptosis, which is associated with up-regulation in p53 expression, phosphorylation at Ser15, and Bak up-regulation.
To confirm that EGCG-induced apoptosis is p53-dependent, we established stable transfectant JB6 cells to knockdown p53 (shp53); shmock stable transfectant JB6 cells were also developed as control.Western blot analysis revealed that f80% p53 was knocked down through stable expression of the shRNA expression construct, targeting p53 in comparison with the control constructtransfected clone (Fig. 2Ca).When the shp53 and shmock expression constructs-transfected cells were treated with 10 to 40 Amol/L EGCG in the presence of 20 nmol/L/mL TPA, cell flow cytometry analysis revealed that silence of p53 lead to substantial suppression of the EGCG-induced apoptosis (Fig. 2Cb).Similarly, when Bak was knocked down through RNAi (90% knockdown; Fig. 2Da), EGCG-induced apoptosis of JB6 cells were attenuated f45% (Fig. 2Db and Dc).These results showed that EGCG-induced apoptosis in JB6 cells is p53-and Bak-dependent.
Overexpression of PP-2Acs prevents EGCG-induced apoptosis and hyperphosphorylation of p53 at Ser-15 in JB6 cells.The Figure 5. Demonstration that p53 directly regulates Bak in JB6 cells.Aa, diagram of the two oligos containing a well-conserved p53 binding site (top ) or mutant p53 binding site (bottom ), which were used for gel mobility shifting assays described in Ab.Ab, gel mobility shifting assays.Nuclear extracts prepared from JB6 cells treated with 40 Amol/L EGCG for 12 h were incubated with g-32 P-ATP-labeled oligo-nucleotide containing wild-type p53 binding site (Aa, top ) under various conditions shown in the figure.The reaction mixtures were then separated with 5% native PAGE.The gel was dried and exposed to X-ray film for 3 h.Lane 1, gel mobility shifting assays with labeled oligo containing wild-type p53 binding site and nuclear extract from JB6 cells after treatment by 40 Amol/L EGCG for 12 h.Lane 2, the same assay as described for lane 1 except that 50-fold of nonlabeled oligo containing the wild-type p53 binding site was added into the reaction.Note that the p53 complex was largely competed off by the nonlabeled oligo.Lane 3, the same assay as described in lane 1 except that the nonlabeled competing oligo containing an mutated p53 binding site (Fig. 5A, bottom ), which could not compete off the p53 complex formed between p53 protein and the oligo containing wild-type p53 binding site.Lane 4, the same assay as described in lane 1 except that the nuclear extracts were preincubated with anti-p53 antibody to remove the p53 protein.Note that when p53 protein was largely precleared, the p53 complex almost disappeared.Ba, diagram to show the luciferase reporter gene driven by mouse bak promoter (À362 to +21) with one copy of well-conserved p53 binding site inserted between À74 to À93.This reporter gene is used in experiments described in Fig. 5Bb.Bb, assays for transient reporter gene expression.JB6 cells were transfected with the Bak-Luc reporter gene (Bak-Luc ) containing one copy of wild-type p53 binding site, or the same construct with the p53 binding site mutated (mBak-Luc ) without or with treatment by TPA (Bak-Luc+TPA and mBAK-Luc+TPA ), or by both TPA plus EGCG (Bak-Luc +TPA +EGCG and mBAK-Luc+TPA+EGCG ), together with an internal control plasmid as described in the Materials and Methods.Twenty-four hours after transfection, these cells were harvested for analysis of luciferase activity as described (34).Note that mutation of the p53 binding site inserted in Bak promoter caused a loss of 50% of the Bak promoter activity and also abolishes the induction by EGCG.Shown here are typical results of three independent experiments.C, dose-dependent response to exogenous p53 by the Bak-Luc promoter with a copy of p53 binding site transfected into JB6 cells (a) and H1299 cells (b).The Bak-luc reporter gene construct and different amount of pCMV-p53 plus the internal control plasmid were introduced into JB6 cell (a) or H1299 cells (b ).Transfection and luciferase activity assays were the same as described in Fig. 5Bb.Note that in the p53(À/À) H1299 cells, lack of the endogenous p53 allows greater dose-dependent response.D, ChIP assay results.The protocols and the oligos used for ChIP assays were described in Materials and Methods.Da, ChIP assay to show that p53 binds to Bak gene p53 binding site.Lane 2, PCR product derived from DNA template immunoprecipitated by normal IgG; lane 3, PCR product derived from 1/50 DNA template immunoprecipitated by anti-p53 antibody; lane 4, PCR product derived from direct input DNA template without immunoprecipitation.A band of 383 bp containing the p53 binding site in Bak gene was amplified.ChIP assay confirms that p53 binds to Bak gene.Db, ChIP assay positive control to show that p53 binds to p21 gene promoter containing a p53 binding site.Lane 2, PCR product derived from DNA template immunoprecipitated by normal IgG; lane 3, PCR product derived from 1 of 50 DNA template immunoprecipitated by anti-p53 antibody; lane 4, PCR product derived from direct input DNA template without immunoprecipitation.A band of 478 bp containing the p53 binding site in p21 promoter was amplified.ChIP assay confirms that p53 binds to p21 promoter.
fact that EGCG negatively regulates PP-2A suggests that PP-2A may be an important target for EGCG to exert its chemopreventive function.To show that this is the case, we stably overexpressed the cDNA encoding PP-2Acs and the corresponding vector as mock.The stable clones of JB6 cells were obtained through G418 selection.Overexpression of exogenous PP-2Acs was confirmed with Western blot analysis (Fig. 3A).When the JB6 cells expressing either the empty pCI-neo vector (Fig. 3Ba) or the PP-2Acs cDNA (Fig. 3Bb) were treated with various concentrations of EGCG, cell flow cytometry analysis revealed that cells overexpressing PP-2Acs displayed distinct ability to resist on apoptosis induced by various concentrations of EGCG in the presence of 20 ng/mL TPA (Fig. 3B-C).
Because EGCG induces p53-dependent apoptosis, the suppression of EGCG-induced apoptosis by PP-2Acs may be derived from its action on p53.To explore this possibility, we examined the changes of the total p53 protein and its phosphorylation status at Ser-15 and other sites induced by EGCG.As shown in Fig. 3D, introduction of a vector into JB6 cells induced detectable p53 expression and its phosphorylation at Ser-15.EGCG treatment of the vector-transfected cells enhanced both p53 level and hyperphosphorylation at Ser-15 in a dose-dependent manner.Phosphorylation at Ser-20 of p53 was not observed (data not shown).Associated with up-regulation of p53 and phospho-p53 at Ser-15, p21, and Bak but not Bax were clearly up-regulated by EGCG in vectortransfected cells.Overexpression of PP-2Acs, however, abolished EGCG-induced up-regulation in p53 expression and phosphorylation at Ser-15, and also prevented up-regulation of p21 and Bak.This result suggests that PP-2A may directly dephosphorylate p53 at the Ser-15 to modulate expression of its downstream genes.
PP-2A directly dephosphorylates p53 at Ser-15 in JB6 cells.To confirm that PP-2A directly dephosphorylates p53 at Ser-15 in JB6 cells, we first conducted in vitro dephosphorylation assay.To do so, we prepared GST and mouse p53 fusion proteins, GST-p53 and GST-p53-S15A, and purified these proteins through glutathione affinity column purification (Fig. 4A).The purified GST-p53 fusion proteins were then labeled with DNA-DPK in the presence of 32 P-g-ATP, and the labeled GST-p53 fusion proteins were used for in vitro dephosphorylation assays.As shown in Fig. 4B, GST-p53 (labeled at Ser-15) fusion protein phosphorylated by DNA-PK in the presence of 32 P-g-ATP can be dephosphorylated by PP-2A.In contrast, p53-S15A was not phosphorylated by DNA-DPK and did not yield detectable free 32 P (Fig. 4B).Next, we conducted coimmunoprecipitation analysis.As shown in Fig. 4C, the proteins immunoprecipitated by anti-PP-2Acs can be detected with anti-p53 antibody (Fig. 4C).Similarly, the proteins immunoprecipitated down by anti-p53 antibody also contains PP-2Acs (Fig. 4C,  3).Finally, we conducted in vivo dephosphorylation assay.As shown in Fig. 4D, the labeled proteins immunoprecipitated by anti-phospho-53 at Ser-15 antibody contains PP-2Acs, which under dephosphorylation condition, released free 32 P from the immunoprecipitated p53.This release is largely inhibited in the presence of specific PP-2A inhibitor (f75% inhibition) and, to a much less degree, by specific PP-1 inhibitor (f25% inhibition).In the presence of 200 nmol/L okadaic acid, the release of free 32 P was almost completely inhibited (Fig. 4D).In contrast, 250 nmol/L CSA, a PP-2B inhibitor, had very little effect (only f7% inhibition).Together, these results suggest that PP-2A is the major phosphatase dephosphorylating p53 at Ser-15 in JB6 cells.
P53 controls Bak gene in JB6 cells.As described above, overexpression of PP-2Acs abolished p53 expression and its hyperphosphorylation at Ser-15.Overexpression of PP-2Acs also prevented up-regulated-expression of the proapoptotic gene, Bak (Fig. 3D).These results suggest that p53 may control Bak in JB6 cells.To explore this possibility, we checked the Bak gene sequence from the gene bank information and found one well-conserved p53 binding site localized at +2635 to +2654, the first intron of the Bak gene.This is not surprising because numerous genes are found containing specific cis-elements in their first introns acting as core elements of enhancers (36,37).An oligo containing this p53 binding site was synthesized (Fig. 5Aa) and used for gel mobility shifting assay.As shown in lane 1 of Fig. 5Ab, a complex was found assembled from the extracted JB6 nuclear p53 and the oligo containing the p53 binding site from Bak gene.This complex band can be significantly competed off with unlabeled wild-type oligo (Fig. 5Ab, lane 2) but could not with unlabeled oligo with the p53 binding site mutated (Fig. 5Ab, lane 3).Moreover, the nuclear extract precleared with an antibody against p53 could no longer bind to the labeled oligo (Fig. 5Ab, lane 4), indicating that p53 indeed interacts with Bak gene p53 binding site.To further confirm that p53 controls Bak in JB6 cells, we conducted three lines of experiments.First, we generated a Bak promoter with a copy of wild-type or mutant p53 binding site (the oligo containing the p53 binding site found in the first intron was inserted at À72 of the Bak promoter) and tested their ability to drive the reporter luciferase gene expression in the absence or presence of EGCG.As shown in Fig. 5Bb, a comparison of the luciferase activities between the Bak promoter wild-type p53 binding site-Luc and the Bak promoter mutant p53 binding site-Luc revealed that presence of a wild-type or a mutant p53 binding site in the mouse bak promoter causes a 54% difference in reporter luciferase activity.Moreover, mutation of the inserted p53 binding site in the bak promoter also abolishes the EGCG-mediated inducibility of the luciferase reporter gene (Fig. 5Bb).Second, we cotransfected the Bak promoter-wildtype p53 binding site-luc and wild-type p53 into JB6 cells, or into H1299 cells lacking functional p53, and examined the dosedependent induction of luciferase activity by exogenous wild-type p53.As shown in Fig. 5C, the dose-dependent induction of the luciferase activity was observed in both types of cells, although lack of the endogenous p53 in H1299 lead to better inducibility.Finally, we conducted ChIP assay to show that p53 directly binds to the Bak gene p53 binding site.As shown in Fig. 5Da, after crosslinking of the EGCG-treated JB6 cells, the anti-p53 antibody was able to precipitate down the complex of p53-Bak gene chromatin fragment containing the p53 binding site.After digestion by protease K, the remaining DNA fragment can be amplified with specific primers for the endogenous enhancer containing p53 binding site.As a positive control, the anti-p53 antibody was also able to precipitate down the complex of p53-p21 promoter sequence containing the p53 binding site (Fig. 5Db).Together, these results show that p53 regulates Bak gene in JB6 cells.
Inhibition of PP-2A activity with okadaic acid enhances EGCG-induced p53-dependent apoptosis.Because up-regulation of PP-2A suppresses EGCG-induced p53-dependent apoptosis, we next examined whether inhibition of PP-2A activity by a specific chemical inhibitor, okadaic acid, would regulate EGCG-induced apoptosis.It is well-established that PP-2A is 100 times more sensitive than PP-1 to okadaic acid in the in vitro assay due to the PP-2A structure features (38).Protein phosphatase activity assayed with directly lysed okadaic acid-treated JB6 extracts revealed that 60 nmol/L okadaic acid can inhibit PP-2A but not PP-1 activity (Fig. 6A ).This conclusion was derived by comparing the phosphatase activity in JB6 cells after treatment by 0.01% DMSO, 60 Amol/L or 200 Amol/L okadaic acid without (lanes 1, 2, and 5) or with addition of thrice of the IC 50 concentrations of the specific inhibitors for either PP-2A (PP2A-I1) or PP-1 (PP1-I2; lane 3, 0.01% DMSO-treated JB6 cell extract plus 90 nmol/L PP2A-I1; lane 4, 60 nmol/L okadaic acid-treated JB6 cell extract plus 90 nmol/L PP2A-I1; lane 6, 0.01% DMSO-treated JB6 cell extract plus 6 nmol/L PP1-I2; lane 7, 200 nmol/L okadaic acid-treated JB6 cell extract plus 6 nmol/L PP1-I2).When PP-2A but not PP-1 was blocked by 60 nmol/L okadaic acid, EGCG-induced apoptosis in the presence of 20 ng/mL TPA in JB6 cells was significantly enhanced (Fig. 6Ba and Bb).To determine whether the enhancement of EGCG-induced apoptosis by inhibition of PP-2A occurs through p53-dependent mechanism, we examined the expression and phosphorylation patterns of p53.As shown in Fig. 6C, even in the absence of EGCG-treatment, inhibition of PP-2A caused slight up-regulation of total p53 expression and distinct enhancement of p53 hyperphosphorylation at Ser-15.Both 10 and 20 Amol/L EGCG moderately enhanced expression of total p53 but distinctly enhanced hyperphosphorylation of p53 at Ser-15 (Fig. 6C).Associated with distinct up-regulation of p53 expression and phosphorylation at Ser-15 under induction by the inhibition of PP-2A and also by EGCG, the expression of p21 and Bak was also up-regulated.These results suggest that PP-2A actively regulates EGCG-induced apoptosis.EGCG-mediated downregulation of PP-2A is an important aspect for EGCG-induced p53-dependent apoptosis.
Figure 6.Inhibition of PP-2A enhances EGCG-induced apoptosis through p53-Bak pathway.A, okadaic acid at a concentration of 60 nmol/L can inhibit PP-2A but not PP-1.PP-2A activity assays in the presence or absence of various inhibitors as indicated in the figure were conducted as previously described (33,34).B, cell flow cytometry analysis.Mouse JB6 Cl41 cells were pretreated with 0.01% DMSO (Ba) or 60 nmol/L okadaic acid (Bb) for 3 h, then further treated with 20 Amol/L EGCG in the presence of 20 ng/mL TPA for 24 h, and finally harvested for analysis of cell flow cytometry as described above.C, inhibition of PP-2A enhances EGCG-induced up-regulation of p53, p21, and Bak protein expression and p53 phosphorylation at Ser-15.JB6 cells were treated as described in Ba and Bb , and then harvested for Wastern blot analysis.Western blot analysis was conducted as previously described (32)(33)(34).Note that inhibition of PP-2A enhances EGCG-induced up-regulation of p5, p21, and Bax expression, and also phosphorylation of p53 at Ser-15.D, schematic diagram to show that PP-2A is an important target regulated by EGCG during its chemopreventive action against carcinogenesis.PP-2A (red ) is actively involved in dephosphorylation of p53 at Ser-15.Dephosphorylation of p53 at Ser-15 favors MDM2-p53 interaction and sensitizes p53 toward ubiquitination and degradation pathway.EGCG negatively regulates PP-2A at the mRNA, protein, enzyme activity levels, as well as localization.As a result, EGCG inhibits PP-2A to promote p53 hyperphosphorylation, which enhances p53 transactivity to up-regulate Bak expression, and thus, promotes p53-dependent apoptosis.The enhancement of p53 transactivity also up-regulates p21 to block CDK/cyclin activity to enhance growth inhibition.Thus, PP-2A plays an important role in mediating EGCG-induced cancer prevention.

Discussion
In the present study, we have shown the following: (a) EGCG at physiologic levels (10 to 20 Amol/L) down-regulates expression of PP-2Acs at protein and enzyme activity levels and also causes change in PP-2Acs localization; (b) EGCG at 10 to 40 Amol/L induces p53-dependent apoptosis of JB6 cells through mitochondrial death pathway; (c) overexpression of PP-2A suppresses p53-dependent apoptosis; (d) PP-2A directly dephosphorylates p53 at Ser-15; (e) P53 controls expression of Bak gene; and ( f ) inhibition of PP-2A activity enhances p53-dependent apoptosis.Together, our results show that EGCG-mediated negative regulation of PP-2A is an important molecular event for the activation of p53-dependent apoptosis during its chemoprevention against cancer (Fig. 6D).
In contrast, during EGCG chemoprevention, whether it acts on protein serine/threonine phosphatases remains largely unknown.In the present study, we show that EGCG at physiologic concentrations (10-20 Amol/L) regulates one of the most important serine/threonine phosphatases, PP-2A.EGCG at 10 Amol/L promotes the nuclear localization of the catalytic subunit of PP-2A.When its concentration is increased to 20 Amol/ L, EGCG clearly down-regulates the protein level and enzyme activity of PP-2Acs.At 40 and 80 Amol/L, EGCG also downregulates the mRNA level for PP-2Acs.A search of the PP-2Acs promoter sequence reveals the presence of multiple cis-elements for AP-1, NF-nB, and several other transcription factors. 5Knowing that EGCG regulates AP-1 and NF-nB (1-3), it is not surprising that EGCG can regulate PP-2Acs mRNA level.
Because PP-2A and PP-1 contributes >90% of total cellular protein serine/threonine phosphatase activities, and PP-2A contributes to f0.3% of the total cellular proteins in eukaryotes (13)(14)(15), it is conceivable that down-regulation of PP-2A by EGCG would have profound consequences.As a major phosphatase, PP-2A may have many different targets.One of such target is the tumor suppressor, p53.We show here that PP-2A can directly dephosphorylate p53 (Fig. 4; see below for more discussion).Overexpression of the catalytic subunit of PP-2A blocks EGCG-induced apoptosis (Fig. 3B-C).Mechanistically, overexpression of PP-2A greatly attenuates p53 hyperphosphorylation at Ser-15, which lead to suppression of EGCG-induced up-regulation of Bak (Fig. 3D).On the other hand, inhibition of PP-2A activity by okadaic acid enhances p53 hyperphosphorylation at Ser-15 and induced elevated expression of Bak (Fig. 6C).Together, these results show that PP-2A is one of the key targets regulated by EGCG in exerting its chemopreventive effects.
PP-2A directly dephosphorylates p53 at Ser-15 to modulate p53 function.Phosphorylation/dephosphorylation is the most important posttranslational modification on p53 functions (40).At the present, 17 serine/threonine residues are found regulated by phosphorylation/dephosphorylation (40).Many laboratories have shown that different signals can activate multiple pathways to phosphorylate p53 at one or more of these serine/threonine residues and, thus, lead to profound changes in stability, transcriptional activity, and apoptotic ability of p53 (40).
Compared with our understanding of p53 phosphorylation by various protein kinases, much less is known about the dephosphorylation of p53.Nevertheless, the few reports have identified the specific phosphatases responsible for dephosphorylating p53 at four specific residues.Long and colleagues (41) have reported that PP-1 may dephosphorylate p53 at Ser-392 in neonatal rat cardiomyocyte.Dohoney and colleagues (42) have shown that in response to DNA damage, protein phosphatases-2A is able to dephosphorylate p53 at Ser-37 in Molt-4 cells.We previously reported that PP-1 dephosphorylates p53 at Ser-15 to promote survival in human lens epithelial cells (43).Recently, we have shown that PP-1 dephosphorylates both Ser-15 and Ser-37 to modulate both transcriptional activity and proapoptotic ability of p53 (33).More recently, Li and colleagues (44) have shown that in U2OS cells, PP-2A is responsible for dephosphorylation of p53 at Thr-55 to activate p53.In the present study, we provide evidence to show that PP-2A can directly dephosphorylate p53 at Ser-15.First, PP-2Acs and p53 can be coimmunoprecipitated.Moreover, in vitro labeled p53 at Ser-15 by DNA-PK in the presence of g-32 P-ATP could be dephosphorylated by purified PP-2A.Furthermore, the pulse-chase labeled p53 when immunoprecipitated with an antibody against phosphorylated p53 at Ser-15 was largely dephosphorylated by the coimmunoprecipitated PP-2Acs.These data indicate that PP-2A can directly dephosphorylate p53 at Ser-15 in JB6 cells.This conclusion is further supported by the fact that overexpression of PP-2Acs causes dephosphorylation of p53 at Ser-15 in JB6 cells (Fig. 3D), and that inhibition of PP-2Acs by okadaic acid leads to p53 hyperphosphorylation at Ser-15 as observed in Fig. 6C.Thus, our results show that in JB6 cells, PP-2A is a major phosphatase that catalyzes dephosphorylation of p53 at Ser-15 residue, which is contrast to what we previously observed in human lens epithelial cells where PP-1 acts as a major phosphatase dephosphorylating p53 at Ser-15 and Ser-37 two residues (Fig. 4; ref. 33).
In the present studies, our results show that dephosphorylation of p53 at Ser-15 by PP-2A has distinct effect on p53 function.We show that p53 can regulate expression of the downstream proapoptotic gene, bak (Fig. 5; see further discussion below).Overexpression of PP-2Acs induces hypophosphorylation of p53 at Ser-15, attenuated expression of bak, and eventual inhibition of EGCG-induced apoptosis (Fig. 3).Our results are consistent with previous studies from numerous laboratories including ours where it was found that dephosphorylation of p53 at Ser-15 and Ser-37 by PP-1 or at Ser-37 and Thr-55 by PP-2A modulates its transcriptional activity on target genes such as Bcl-2, Bax, and p21 (33,(41)(42)(43)(44).In the present study, we also observed that dephosphorylation of p53 at Ser-15 by PP-2A attenuates p21 expression, which is associated with EGCG-mediated inhibition of cell growth (1-3).
P53 directly controls expression of the proapoptotic regulator, Bak, to regulate apoptosis.The tumor suppressor, p53, is a master regulator of apoptosis in many types of cells (40).As a master regulator of apoptosis in different cells, p53 regulates apoptosis by two mechanisms.First, it acts as a transcriptional factor to regulate expression of many genes involved in apoptosis.One set of such genes are the ones coding for members of the Bcl-2 family including Bcl-2 and Bax (45).Second, p53 could be translocated into mitochondria where it antagonizes the antiapoptotic ability of Bcl-2 and Bcl-XL (46).Although it has been shown that p53 can activate Bak in mitochondria to induce apoptosis (47) and EGCG induces up-regulation of both p53 and Bak (48), it remains to be determined whether p53 regulates Bak gene expression.In the present study, we present several lines of evidence to show that p53 controls expression of bak, another important p53 downstream gene mediating apoptosis.First, analysis on the bak gene sequence reveals the presence of a wellconserved p53 binding site located from +2635 to +2654, in the first intron of the Bak gene.Gel mobility shifting assays reveal that a nuclear protein can strongly bind to this conserved p53 binding site.When the nuclear extracts were precleared with an anti-p53 antibody, the precleared nuclear extract lost most of the ability to bind to the bak gene oligo containing the p53 binding site.Second, when a luciferase reporter gene driven by the bak gene promoter inserted with a copy of the wild-type p53 binding site or a copy of mutant p53 binding site is transfected into JB6 cells, the reporter gene activity assays reveal that mutation of the p53 binding site causes distinct reduction of the reporter luciferase activity (Fig. 5Bb).Mutation of the p53 binding site in the Bak-Luc construct also abolishes, to a large degree, the inducing ability of the reporter gene by EGCG.Third, cotransfection of this Bak promoter-wild-type p53 binding site-Luc reporter gene with wild-type p53 expression construct induces a dose-dependent increase in luciferase activity as tested in both JB6 and H1299 cells.Finally, ChIP assays confirmed that p53 binds to Bak gene p53 binding site.Together, our results show that p53 regulates Bak expression in mouse JB6 skin cells.Considering that p53 can directly activate Bak in mitochondria to induce apoptosis (47), our demonstration that p53 also controls Bak expression suggest that the p53-Bak pathway plays an important role in EGCG-mediated chemoprevention of JB6 cells.This is further supported by the fact that knockdown of Bak in JB6 cells caused a 45% attenuation of the EGCG-induced apoptosis (Fig. 2D).Moreover, EGCG regulation of PP-2A also conveys the signaling pathway to p53-Bak (Fig. 6D).
The molecular mechanisms by which PP-2A regulates carcinogenesis.As mentioned above, PP-2A exists in cells in two major forms: holoenzyme and core enzyme (16,22,38).The core enzyme consists of a 36-kDa catalytic C subunit and a 65-kDa regulatory A subunit (22).The holoenzyme is composed of a core enzyme to which one of several regulatory B subunits is bound (22).The A and C subunits exist in two isoforms: Aa and Ah, and Ca and Ch, respectively.The B subunits fall into four subfamilies, designated B, B', B'', or B''', which seem unrelated by sequence alignment.The B family has four members: Ba, Bh, Bg, and By.The B ¶ family consists of five genes encoding B'a, B'h, B'g, B'y, and B'q as well as several isoforms and splice variants.The B'' family has four members, designated PR48, PR59, PR72, and PR130.The latter two are splicing variants of the same gene.The B''' family has two members: striatin and SG2NA (19).
Both A and B subunits of PP-2A are implicated in carcinogenesis.For the scaffold A subunit, it was found that the gene encoding the Ah subunit of PP-2A is mutated or deleted in 15% of primary lung and colon cancers (24).Calin and colleagues (25) have reported that both the Aa and Ah subunit isoforms are genetically altered in a variety of primary human cancers.These results suggest that PP-2A seems to act as a tumor suppressor.Based on the location of the mutated amino acids in the intrarepeat loops or nearby in Aa or Ah, it has been suggested that the mutant A subunits are likely defective in binding B and/or C subunits (19,26,27).As a result, the specific PP-2A activity needed in these tissues is diminished and the normal functions of certain targets or signaling pathways are interrupted.For example, PP-2A is colocalized with Bcl-2 in mitochondria to modulate Bcl-2 function.Lack of the functional scaffold A subunits may lead to absence or insufficiency of the mitochondrial PP-2A activity and, thus, favor the Bcl-2 hyperphosphorylation at Ser-70 (49) and inhibition of cancer cell apoptosis, allowing accumulation of cancer cells for tumor formation.On the other hand, mutation of the scaffold A subunits would leave more unbound catalytic subunit of PP-2A, a situation similar to overexpression of PP-2Acs.The free PP-2Acs could move into nucleus and negatively regulates p53 function through dephosphorylation as we have shown in the present study, leading to attenuation of p53-dependent apoptosis and further enhancement of tumor development.
Certain members of the regulatory B, B', B'', or B''' family subunits are also involved in regulation of cancer development.In this regard, numerous previous studies have shown that PP-2A interacts with the SV40 small antigen (ST), and such interaction is essential for transformation of certain human cells.Yu and colleagues (28) reported that the transformation of primary human diploid fibroblasts and of mesothelial cells in culture depends on both SV40 large T and small T.Moreover, they found that inhibition or alteration of PP-2A by SV40 small antigen is required for cell transformation.Similar results were reported by Hahn and colleagues (29) using human fibroblasts and human embryonic kidney cells.At the molecular level, the SV40 small antigen displaces the B family subunits of PP-2A and binds to both A and C subunits to inhibit PP-2A activity (22).The importance of the B family members in tumor suppression is further revealed by a recent study where Chen and colleagues (50) have discovered that suppression of the B family regulatory subunit, B56g, inhibits PP-2A-specific phosphatase activity in human embryonic kidney epithelial cells immortalized by SV40 large T antigen, hTERT, and H-RAS and confers these cells anchorage-independent growth and, thus, leads to formation of tumors.
Although accumulated evidence suggests that both A and B subunits of PP-2A are involved in regulation of carcinogenesis, it remains to be determined if the catalytic subunit of PP-2A is directly involved in regulation of cancer development.In the present study, we present evidence to show that the catalytic subunit of PP-2A is also involved in regulation of carcinogenesis.Overexpression of PP-2Acs blocks EGCG-induced apoptosis, which is mediated through the p53-Bak pathway.Inhibition of p53-dependent apoptosis could interrupt the dynamic homeostasis between cell proliferation and cell death, thus promoting cell transformation.Indeed, our unpublished results suggest that overexpression of the catalytic subunit for PP-2A promotes cell transformation.

Figure 3 .
Figure3.Overexpression of PP-2Acs blocks EGCG-induced apoptosis, p53 up-regulation and hyperphosphorylation, and p21 and Bak up-regulation.A, Western blot analysis to determine the PP-2Acs levels in JB6 C141 cells transfected with the pCI-neo vector (left ) or pCI-PP-2Acs (right ).Establishment of the stable expression clones, pCI-neo-JB6 and pCI-PP2Acs-JB6, was described in the Materials and Methods.B, cell flow cytometry of both vector-(Ba) and pCI-PP-2Acs (Bb)-transfected clones were treated with 20 Amol/L EGCG, as described in Fig.1B, for 24 h and then harvested for cell flow cytometry as previously described(32,33).C, quantitative comparison of EGCG-induced apoptosis in the vector-transfected cells (vector ) or PP-2Acs-transfected cells (PP-2Acs ).Shown here are typical results of three independent experiments.D, overexpression of PP-2A blocks EGCG-induced up-regulation of p53, p21, and Bak proteins, and p53 phosphorylation at Ser-15.Both pCI-neo-JB6 and pCI-PP-2Acs-JB6 cells were treated as described in Fig.1Band then harvested for Wastern blot analysis as previously described(32)(33)(34).Note that overexpression of PP-2Acs attenuates EGCG-induced up-regulation of p53, p21, and Bak protein levels, and p53 phosphorylation at Ser-15.Shown here are typical results of three independent experiments.

Figure 4 .
Figure 4. PP-2A dephosphorylates p53 at Ser-15 in JB6 cells.A, the SDS-PAGE analysis of the fusion proteins for GST-p53 and GST-p53-S15A before (left ) and after (right ) glutathione column purification.B, in vitro dephosphorylation assays by PP-2A.The two substrates, GST-p53 and GST-p53-S15A, were labeled with DNA-PK and precipitated by TCA after labeling.Then equal amount of each protein (2 Ag protein for each sample) was mixed with 1Â PP-2A dephosphorylation buffer and 10 ng PP-2Acs (obtained from Calbiochem, Inc.) in the absence or presence of specific PP-2A inhibitor, PP2A-I1.The mixture was incubated 10 min at 30jC.After the reaction, the released free 32 P was counted.The final results were averaged from three independent experiments in which the free 32 P released from each dephosphorylation reaction was recorded.C, reciprocal immunoprecipitation (IP ) to show that PP-2Acs and p53 form interacting complex.JB6 cells were harvested for extraction of total proteins, which were used for immunoprecipitation with anti-PP-2Acs antibody (1 and 2 ) or anti-total p53 antibody (3 and 4 ).The immunoprecipitated samples were then subjected to Western blot analysis as described before(32)(33)(34).The supernatant represents the sample after immunoprecipitation.Shown here are typical results of three independent experiments.D, in vivo dephosphorylation of p53 at Ser-15 by PP-2A.JB6 cells were labeled with [ 32 P]-P i (200 ACi/mL) in phosphatase-free DMEM for 30 min and then subject to 80 Amol/L EGCG treatment for another 4 h in the presence of the [ 32 P]-P i (200 ACi/mL).Total proteins were extracted for immunoprecipitation with an antibody against phospho-p53 at Ser-15.The immunoprecipitated protein complexes were mixed with phosphatase reaction buffer in the presence or absence of various inhibitors and then incubated at 30jC for 10 min.After TCA precipitation, the supernatant fraction was recovered for counting the release of free 32 P in a scintillation counter.Immunoprecipitated sample with antiactin antibody was also used for the parallel dephosphorylation assay (mock ).The dephosphorylation assays were conducted in the absence (extract ) or presence of PP-1 inhibitor, PP1-I2 (Extract +PP1-I2 ) or 200 nmol/L okadaic acid (Extract +OA ), PP-2A inhibitor, PP2A-I1 (Extract +PP2A-I1 ), and PP-2B inhibitor, 250 nmol/L cyclosporin A (Extract +CSA ).The results shown here are averaged from three independent experiments.Note that the inhibitor blocking PP-2A but not PP-2B was able to inhibit f75% dephosphorylation.In contrast, the specific inhibitor for PP-1 only inhibited f25% of dephosphorylation, indicating that in JB6 cells, PP-2A is the major phosphatase that dephosphorylates p53 at Ser-15.IB, immunoblot.