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Regulation of the human AP-endonuclease (APE1/Ref-1) expression by the tumor suppressor p53 in response to DNA damage

Regulation of the human AP-endonuclease (APE1/Ref-1) expression by the tumor suppressor p53 in... Published online 21 January 2008 Nucleic Acids Research, 2008, Vol. 36, No. 5 1555–1566 doi:10.1093/nar/gkm1173 Regulation of the human AP-endonuclease (APE1/Ref-1) expression by the tumor suppressor p53 in response to DNA damage 1,2 3 3 1 Amira Zaky , Carlos Busso , Tadahide Izumi , Ranajoy Chattopadhyay , 2 1 1, Ahmad Bassiouny , Sankar Mitra and Kishor K. Bhakat * Department of Biochemistry and Molecular Biology, Sealy Center for Molecular Medicine, University of Texas Medical Branch, TX-77555, Galveston, USA, Department of Biochemistry, Alexandria University, Alexandria, Egypt, 21511 and Department of Otolaryngology, Louisiana State University Health Science Center, LA 70112, New Orleans, USA Received October 31, 2007; Revised December 19, 2007; Accepted December 20, 2007 role in the base excision repair (BER) pathway for ABSTRACT damaged bases induced by reactive oxygen species and The human AP-endonuclease (APE1/Ref-1), an alkylating agents, and for abasic (AP) sites generated essential multifunctional protein, plays a central after excision of oxidized and alkylated bases by DNA role in the repair of oxidative base damage via glycosylases (1–3). As an endonuclease, APE1 cleaves the 0 0 the DNA base excision repair (BER) pathway. The AP site to generate 3 -OH and 5 -deoxyribose phosphate mammalian AP-endonuclease (APE1) overexpres- termini, and the 3 -OH terminus is utilized by a DNA sion is often observed in tumor cells, and confers polymerase, usually DNA polymerase b, for repair synthesis in mammalian cells (4). APE1 was independently resistance to various anticancer drugs; its down- identified as a reductive activator of the c-Jun transcrip- regulation sensitizes tumor cells to those agents tion factor in vitro, and named Ref-1 (5). Subsequently, via induction of apoptosis. Here we show that wild several other transcription factors (including p53, type (WT) but not mutant p53 negatively regulates hypoxia-inducible factor and NF-kB) were also found to APE1 expression. Time-dependent decrease was be activated by APE1 (6–8). In addition, APE1 directly observed in APE1 mRNA and protein levels in the 2+ acts as a trans-acting factor by binding to negative Ca (+/+) human colorectal cancer line HCT116 p53 , but 2+ response elements (nCaRE) during Ca -dependent not in the isogenic p53 null mutant after treatment repression of the human parathyroid hormone and renin with camptothecin, a DNA topoisomerase I inhibitor. genes (9–11). Furthermore, ectopic expression of WTp53 in the Given its multiple functions, it is not surprising that p53 null cells significantly reduced both endogen- APE1 expression is regulated in vivo in a complex manner. ous APE1 and APE1 promoter-dependent luciferase We were one of the first to show the activation of APE1 gene by oxidative stress; several other laboratories sub- expression in a dose-dependent fashion. Chromatin sequently confirmed our observation (12–14). Moreover, immunoprecipitation assays revealed that endogen- based on the identification of the nCaREs in the APE1 ous p53 is bound to the APE1 promoter region that promoter itself, we suggested that APE1 regulates its own includes a Sp1 site. We show here that WTp53 expression (15). While cell cycle-dependent expression of interferes with Sp1 binding to the APE1 promoter, APE1 was observed in NIH3T3 cells (16), the basal which provides a mechanism for the downregulation expression of APE1 is variable. The increased APE1 level of APE1. Taken together, our results demonstrate is often observed in tumor cells including prostate, that WTp53 is a negative regulator of APE1 expres- osteosarcomas, lung and cervical carcinomas compared sion, so that repression of APE1 by p53 could to normal tissues (17–20). In other types of cancer, provide an additional pathway for p53-dependent subcellular localization of APE1 is altered relative to induction of apoptosis in response to DNA damage. that in normal tissues (21–23). Tumor cells often over- express APE1 and are resistant to chemotherapeutic drugs and ionizing radiation (19,24,25). In contrast, INTRODUCTION siRNA-mediated downregulation of APE1 induced apop- The mammalian AP-endonuclease (APE1) is a ubiquitous tosis of many tumor cell types and enhanced cell and remarkably multifunctional protein. It plays a central sensitization to ionizing radiation and chemotherapeutic *To whom correspondence should be addressed. Tel: 409 772 1779; Fax: 409 747 8608; Email: [email protected] 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 1556 Nucleic Acids Research, 2008, Vol. 36, No. 5 agents (26–29). We recently showed that APE1 inactiva- MATERIALS AND METHODS tion-induced apoptosis in mouse embryo fibroblasts Cell culture and treatment (MEF) conditionally nullizygous for endogenous APE1, The human colorectal adenocarcinoma lines, HCT116 an effect that could be prevented by ectopic expression of (+/+) (/) p53 with WTp53 and p53 null HCT116 p53 human APE1 (29). Using a complementation assay, we (a gift from Dr B. Vogelstein, Johns Hopkins University also showed that both the repair and transcription School of Medicine) were grown in McCoy’s 5A (Gibco regulatory functions of APE1 are required to prevent Life Technologies, Carlsbad, CA, USA) medium supple- apoptosis of MEF (29). Thus, elucidating the molecular mented with 10% fetal bovine serum (Sigma-Aldrich, mechanisms controlling APE1 expression has profound St Louis, MO, USA), 100 U/ml penicillin and 100mg/ml implications for cancer therapy from both basic and streptomycin (Gibco BRL, Carlsbad, CA, USA) in a 5% clinical perspectives. C0 incubator at 378C. Cells were treated with 200 nM The tumor suppressor gene p53, dubbed ‘the guardian camptothecin (CPT) (Sigma, St Louis, MO, USA) and/or of the genome’, encodes a sequence-specific transcription 30mM pifithrin-a (PFT-a) (Sigma, St Louis, MO, USA). factor that activates a variety of cellular genes in response to DNA damage, hypoxia and other stress signals (30,31). Plasmids and luciferase assays p53 is one of the most commonly mutated genes in human cancers; 50% of all human cancers lack the wild type Generation of APE1 promoter–reporter plasmids contain- (WT) p53 gene allele, and these tumors respond poorly ing DNA fragments (4800/+65) and (1800/+65) of to chemotherapy (32,33). In response to DNA damage or the 5 regulatory region was described earlier (15). Other other cellular stresses, p53 is activated and induces cell reporter plasmids with various lengths of the APE1 5 regulatory region were generated by PCR and cloned into cycle arrest or apoptosis, depending on the severity of the the luciferase reporter vector pGL3 (Promega, Madison damage and the context of the cell cycle, and thus helps (/) WI, USA) using standard procedures. HCT116 p53 to maintain genomic stability and prevents cancer (34). cells were cotransfected with 500 ng of the APE1-reporter Once activated, p53 has the ability to arrest cell cycle by plasmids and expression plasmids for WT or mutant transactivation of Waf-1/p21, 14-3-3d etc., or induce cells [Val143Ala (V143A) and L22G, T23S] p53 or equivalent to undergo apoptosis by both transcription-dependent and amounts of empty vector using LipofectAMINE 2000 -independent mechanisms (35–37). p53 activates many (Invitrogen, Life Technologies, Carlsbad, CA, USA), downstream target genes involved in the mitochondrial according to the manufacturer’s instructions. At 48 h signaling pathway during apoptosis including BAX, after the transfections, cells were lysed with the reporter PUMA and the death receptor (38–40). In addition, p53 lysis buffer (Promega), and the luciferase activity in the has been shown to downregulate many genes, including cell lysates was measured in a luminometer (AutoLumant cell survival genes such as survivin, bcl-2, IGF1R and DNA LB593, Berthold, Oak Ridge, TN, USA) by the luciferase repair gene O -methylguanine-DNA-methyltranferase assay kit (Promega, Madison, WI, USA) according to the (MGMT) with the net result of facilitating apoptosis manufacturer’s protocol. The luciferase activity was induction (41–46). normalized to the amount of protein in the lysate. Potential link between APE1 expression and p53 status in various tumors and their resistance to chemotherapeu- Preparation of total cell extract and western blot analysis tic drugs have been documented (24,47). Despite several studies showing the involvement of APE1 and p53 in drug HCT116 cells were lysed in a lysis buffer (50 mM Tris- resistance induction in tumor cells, the question of HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% TritonX- whether p53 regulates APE1 expression after genotoxic 100, and protease inhibitor cocktail (Roche, Nutley NJ, stress has not been addressed. Elucidating the direct role USA) as described earlier (48). Whole-cell extracts (25 mg) of p53 on APE1 gene expression in human cells should be were subjected to 12.5% SDS–PAGE and transferred to a of major clinical significance, and a clear understanding of nitrocellulose membrane (Trans-Blot 0.2mm, Bio-Rad, Hercules, CA, USA). Western immunoblotting analyses the mechanisms by which p53 controls APE1 expression with mouse monoclonal antip53 antibody (sc-DO-1, could help in effective use of chemotherapeutic agents in dilution 1:200, Santa Cruz Biotechnology, CA, USA), the treatment of tumors expressing WT or mutant p53. rabbit polyclonal antiAPE1 (dilution 1:2000), or rabbit In this study, we show that activation of p53 after geno- monoclonal antib-actin antibody (Sigma, 1:1000 dilution) toxic stress downregulates APE1 expression in HCT116 (+/+) were carried out using enhanced chemiluminescence assay p53 human colon carcinoma cells, but not in the p53 (ECL kit, Amersham, London, UK). null mutants derived there from. Transgenic expression of WTp53 in p53 null cells downregulate both endogenous RNA isolation and RT-PCR analysis APE1 level and expression of a reporter whose activity is dependent on APE1 promoter activity. We also present Total RNA was extracted using Rneasy Mini kit evidence that p53 is associated with the endogenous APE1 (QIAGEN) according to the manufacturer’s protocol. promoter in vivo, and that interference of specificity For quantitative, real-time RT-PCR, one step RT-PCR protein (Sp1) binding to the APE1 promoter by WTp53 was performed with 100 ng of cellular RNA for both the and subsequent recruitment of histone deacetylase target gene and an endogenous control in singleplex tubes (HDAC) to the promoter could explain downregulation using TaqMan one-step RT-PCR master mixture reagent of APE1 by WTp53. kit (P/N 4309196). TaqMan MGB probes (FAMTM Nucleic Acids Research, 2008, Vol. 36, No. 5 1557 dye-labeled) for APEX1 gene and 18s rRNA (VICTM-dye Dynamics, Piscataway, NJ, USA) and analyzed using labeled probe) TaqMan assay reagent (P/N 4319413E) IMAGEQUANT software. for endogenous control (assay-on-DemandTM (P/N 4331182) were used. The cycling parameters in an ABI 7000 (Applied Biosystem, Foster City, CA, USA) thermal cycler were as follows: reverse transcription at 488C for RESULTS 30 min. AmpliTaq activation 958C for 10 min, denatura- WTp53 downregulates APE1 mRNA and protein levels tion 958C for 15s, and annealing/extension 608C for 1 min. We examined the effect of WTp53 on APE1 expression The amount of target was calculated after normalization (+/+) (/) in HCT116 p53 and HCT116 p53 cell lines. To to the 18s RNA as an endogenous control. activate p53, these cells were treated with CPT, a DNA Chromatin immunoprecipitation (ChIP) assay topoisomerase I inhibitor, and total RNA was isolated (+/+) (/) at the indicated times for quantitation of APE1 mRNA HCT116 p53 and HCT116 p53 cells were treated levels by real-time RT-PCR. We observed a significant with CPT at final concentration of 200 nM for 6 h unless decrease in the APE1 mRNA level at 18 h after treatment otherwise indicated. Cells were washed twice with ice-cold with CPT with simultaneous activation of WTp53 in PBS, fixed with 1% formaldehyde for 10 min at RT, (+/+) HCT116 p53 cells (Figure 1A). Treatment with CPT washed twice with cold PBS, and harvested in cell scraping also reduced APE1 mRNA level in p53-negative HCT116 solution (1X PBS and 0.5 mM PMSF). Pellets were (/) p53 cells, although not to the same extent as in the collected by centrifugation at 1200 rpm for 10 min at 48C (+/+) p53 cells indicating a relationship between the p53 and resuspended in ice-cold lysis buffer (Active Motif, status and the downregulation of APE1 expression. ChIP-IT, Carlsbad, CA, USA, supplemented with 0.5 mM Treatment with CPT also induced the p21 level in PMSF and 0.5 mM protease inhibitor cocktail), and then (+/+) HCT116 p53 cells concomitant with enhanced p53 incubated on ice for 30 min. The DNA fragments were levels (Figure 1B). Activation of the p21 gene was used to digested with mung bean nuclease to an average size of monitor in vivo function of p53. Although a transient 200–500 bp, as empirically estimated by agarose gel increase in the APE1 protein level was observed soon after electrophoresis of the digest. Immunoprecipitations were (1 h) CPT treatment, consistent with the decreased APE1 performed with antip53 (DO-1), IgG (as a negative mRNA level, the APE1 polypeptide level was also control) and antiSp1 (as a positive control) antibodies. (+/+) significantly reduced in HCT116 p53 cells at 18 h After sequential washing, DNA–protein complexes were after treatment with CPT (Figure 1B, right panel). eluted with elution buffer (1% SDS, 0.1 M NaHCO , We further confirmed the role of p53 by treating 0.01 mg/ml herring sperm DNA). The crosslinks were cells with the water soluble p53 inhibitor PFT-a, which reversed by heating at 658C overnight, and treatment with blocked activation of p53-regulated genes, including proteinase K (0.17mg/ml) for 1 h, and the DNA then cyclin G, p21/Waf-1 and mdm2, and also inhibits isolated using a column (Active Motif, ChIP-IT) accord- apoptosis (49–51). Treatment with PFT-a attenuated ing to the manufacturer’s guidelines. Recovered DNA was (+/+) APE1 repression after CPT treatment in p53 cells suspended in 50ml of TE. PCR amplification of DNA was carried out with diluted aliquots using appropriate (Figure 1C), while it had no effect on the APE1 level in (/) primers to generate PCR products spanning +24 to p53 cells (Figure 1D). Taken together, these results 368 bp and 118 to 253 bp of the APE1 promoter suggest that downregulation of APE1 expression is region. The PCR products were separated by 1.5% p53-dependent. agarose electrophoresis in Tris-borate-EDTA buffer and To further confirm that WTp53 acts as a negative stained with ethidium bromide. regulator of APE1 expression, we investigated the effect of WTp53 overexpression on the APE1 protein and mRNA Electrophoretic mobility shift assay (EMSA) levels in HCT116 p53 null cells. Various amounts of expression plasmid for WTp53 were used for transfection Electrophoretic mobility shift analyses were performed of p53 null cells, and the expression of p53 and p21 was as described earlier (38) with some modifications. The 0 32 0 confirmed by western analysis of the cell extracts 5 P-labeled oligo 5 -AGAGAGGGAGGCGAGGCTA (Figure 2A). Overexpression of p53 decreased APE1 AGCGTCTCCGTCACGT-3 (potential p53-binding site mRNA and protein levels in a dose-dependent manner on APE1 promoter) and 5 -TTG AAC ATG TCC CAA (Figure 2A and B). To confirm that ectopic p53 was CAT GTT GA-3 containing the p53 consensus sequence transcriptionally active, we measured the p21 levels in from the human p21 promoter were annealed with nuclear extracts of p53-transfected cells (Figure 2C), and appropriate complementary stands to generate duplex its binding to double-stranded oligonucleotides containing oligos. The DNA (50 fmol) was then incubated with p53 (/) the p53-binding motif of the p21 promoter. Figure 2C or nuclear extracts (5mg) from HCT116 p53 cells for shows predominant localization of p53 in nuclear fraction 20 min at 258C in a buffer containing 40 mM HEPES- of cells after ectopic expression of WTp53. EMSA of the KOH, pH 7.5, 50 mM KCl, 1 mM MgCl , 0.5 mM EDTA, same extract confirmed the presence of WTp53 which 0.5 mM DTT, 10% glycerol, 1 mg of poly dI-dC (Sigma). formed a shifted complex with the p53 cis sequence After electrophoresis in non-denaturing 5% polyacryla- mide gels in Tris-borate buffer at 48C, the gels were (Figure 2D). Together, these results further confirm that dried and exposed to PhosphorImager (Molecular p53 acts as a repressor of APE1 expression. 1558 Nucleic Acids Research, 2008, Vol. 36, No. 5 1.4 1.2 HCT116 p53 (+/+) HCT116 p53 (−/−) 0.8 0.6 0.4 0.2 c 1h3h6h 18h Time (h) 0 1 3 6 18 24 p53 APE1 p21 b-actin CPT (200 nM) −−++ PFT-a (30 µM) −− ++ p53 APE1 p21 b-actin CPT (200 nM) − + − + p53 PFT-(30 µM) −− ++ APE1 b-actin (+/+) Figure 1. Repression of APE1 gene expression in HCT116 p53 cells after CPT treatment. (A) Real-time RT-PCR analysis of APE1 mRNA (+/+) (/) from HCT116 p53 and HCT116 p53 cells at indicated time points after CPT treatment. Results correspond to mean  SD from three (+/+) separate experiments. (B) After treating HCT116 p53 cells with CPT (200 nM), as described under Materials and Methods section, total lysates of cells harvested at the indicated times were analyzed for p53, APE1 and p21 levels by western blotting. b-actin was used as the loading control. Right panel, graphical representation of APE1 protein level (normalized to b-actin) at indicated time points after CPT treatment. Results correspond (+/+) (/) to mean  SD from three separate experiments. (C and D) HCT116 p53 or p53 cells were treated with CPT (200 nM) for 24 h in the presence or absence of the p53 inhibitor PFT-a and total lysates were analyzed for p53, APE1 and p21 levels by western blotting. b-actin was used as the loading control. Right panel, graphical representation of APE1 protein level (normalized to b-actin) after treatment with CPT or PFT-a. Results correspond to mean  SD from three separate experiments; P< 0.05. APE1 promoter activity is repressed by WTp53, but not dose-dependent decrease in APE1 promoter activity (up to by mutant p53 250-fold compared to the vector control, Figure 3A). Increase in p53 expression was confirmed by western To determine whether p53-mediated downregulation analysis of the same cells extract with antip53 antibody of APE1 occurred at the promoter level, we exam- (Figure 3B). Because p53 is an activator of the p21 gene, ined the effect of p53 overexpression on APE1 promo- we simultaneously examined the effect of ectopic expres- ter-dependent luciferase activity in a transient reporter sion of p53 on p21 promoter-dependent luciferase activity. expression assay. We cotransfected an APE1 promoter luciferase reporter construct (4000/+65 APE1-Luc, The increase in p21 promoter-dependent luciferase activity containing the APE1 promoter sequence from 4000 to with increasing amounts of input p53 plasmid indicated +65 bp) and the WTp53 expression plasmid into HCT116 that the inhibitory effect of p53 on APE1 promoter was (/) p53 cells. Ectopic expression of WTp53 induced a not due to general inhibition of transcription (Figure 3C). (+/+) HCT116 p53 (−/−) (+/+) HCT116 p53 HCT116 p53 APE1 mRNA level (normalized to 18s) Nucleic Acids Research, 2008, Vol. 36, No. 5 1559 WTp53 1.2 plasmid (mg) mock 0.3 0.6 1.2 p53 0.8 0.6 APE1 0.4 0.2 p21 b-actin mock 0.3 0.6 1.2 WTp53 plasmid (mg) B 2 1.8 (−) PFT-a 1.6 1.4 (+) PFT-a 1.2 0.8 0.6 * 0.4 * 0.2 WTp53 plasmid (mg) --- 0.3 0.6 1.2 CD (−/−) p53 nuclear extract − ++++++ Empty vector − −− 0.6 −− 0.6 WTp53 WTp53 plasmid 0.3 0.6 0.6 plasmid (mg) − −−− mock 0.3 0.6 Anti-p53 antibody − − −−− + + p53 Lamin B p21 Figure 2. Decreased APE1 protein and mRNA levels after ectopic expression of WTp53 in p53 null cells. (A) Total cell extracts were analyzed for p53, APE1 and p21 levels by western blotting. b-actin was used as the loading control. Right panel, graphical representation of APE1 protein level (normalized to b-actin) after transfection with various amounts of WTp53 expression plasmid. Results correspond to mean  SD from three separate (/) experiments. (B) Real-time RT-PCR analysis of APE1 mRNA levels in HCT116 p53 cells transfected with various amounts of WTp53 expression plasmid and incubated with PFT-a or vehicle for 48 h. Results correspond to mean  SD from three separate experiments. (C) Western analysis of p53 and p21 levels in nuclear fraction in p53 null cells after ectopic expression of WTp53. Lamin B was used for nuclear extracts loading control. (D) EMSA of nuclear extracts used in (C) using P-labeled duplex oligo from p21 promoter containing consensus p53-binding sequence; P< 0.05. (/) Moreover, we observed that CMV promoter-dependent effect (Figure 3D). Western analysis of p53 cells b-galactosidase and thymidine kinase promoter-dependent lysates transiently transfected with p53 expression plas- renilla-luciferase (pRL-TK, Promega) levels were also mids revealed that the p53 mutant polypeptides were more stable than the WT protein, and were present at higher increased due to overexpression of WTp53 for unknown levels (2- to 3-fold, Figure 3E). These results indicate that reasons (data not shown). We thus could not therefore use despite these higher levels, mutant p53 could not inhibit these reporter plasmids for normalizing transfection the APE1 promoter activity. efficiency. In any case, our results indicate that the p53- dependent repression is APE1 promoter specific, and Identification of p53-responsive cis element(s) not a general phenomenon. We also examined the effects in APE1 promoter of two p53 point mutants on APE1 promoter activity namely, V143A, a missense mutation which inactivates To identify the cis element(s) responsible for p53-mediated p53’s sequence-specific DNA binding, and a Leu22Gln downregulation of the APE1 promoter, we carried out Trp23Ser (22,23), double mutant which retains the ability promoter deletion analysis. We have shown earlier that to bind p53-cis sequences but lacks transcriptional the APE1 promoter contains multiple negative regulatory activation ability (52). Overexpression of WTp53 signifi- and enhancer elements, including two nCaRE-B elements cantly decreased luciferase activity under the control of upstream (4000 to 1800 bp) of the basal promoter (15). the APE1 promoter (reaching up to 60-fold with 50 ng of To test whether p53-induced repression of APE1 promoter WTp53), while the mutants had no significant inhibitory activity is mediated through the cis elements located APE1 mRNA level (normalized to 18s) Fold change in APE1 protein level 1560 Nucleic Acids Research, 2008, Vol. 36, No. 5 (/) A in this region, we cotransfected HCT116 p53 cells with WTp53 and the reporter plasmid containing 1800 to +65 bp (1800/+65 APE1-Luc) of the APE1 promo- ter. Deletion of the promoter sequence from 4000 to 1800 bp significantly increased the basal promoter activity (9-fold, Figure 4A), confirming our earlier observation about the presence of negative regulatory elements within this sequence (15). Furthermore, ectopic expression of WTp53 caused a decrease in the promoter (1800/+65 APE1-Luc) activity in a dose-dependent −4000/+65 APE1-Luc (0.5µg)++ + + + + + + WTp53 (mg) -- 0.01 0.03 0.05 0.07 0.1 0.3 0.5 manner (Figure 4B). We then carried out detailed mapping of the p53 regulatory sequence by using a series of 5 promoter p53 B deletion constructs. DNA fragments of the 5 regulatory region (shown in Figure 5A) were cloned upstream of the b-actin luciferase coding region and their promoter activity was determined by cotransfection as before. All deletion constructs except 7 and 8 showed basal promoter activity 3.5 that was abolished by deleting residues 143 to 118 (Figure 5A and B) indicating that the sequence upstream of 118 is required for basal promoter activity. Although 2.5 deletion of 4000 to 1800 decreased p53-mediated downregulation of promoter activity by 1.5-fold, lucifer- 1.5 ase activity of all the deletion constructs was significantly downregulated after cotransfection with WTp53 (Figure 5B). However, elimination of the sequence from 0.5 184 to 143 (construct number 6, Figure 5A) abolished the inhibitory effect of p53 (Figure 5B), indicating that the cis element involved in p53-dependent repression is localized within this sequence. Furthermore, deletion of WTp53 (ng) --- 10 50 the +65 bp region downstream to the transcription start site (construct number 5, Figure 5A) had no effect on p53- mediated repression, indicating that these sequences are * * not required for repression (Figure 5B). Thus the p53- APE1-Luc responsive cis elements appeared to be located within PRC-cmv WTp53 the proximal region (184 to 143 bp) of the APE1 143-p53 22.23-p53 promoter. Lack of direct binding of p53 to the APE1 promoter in vitro To identify the putative p53-responsive cis element(s) in the 41 bp (184 to 143) APE1 promoter sequence, we performed a computer search for potential transcription factor binding sites. Putative binding sites were identified one each for Sp1 and upstream factor (USF); however, no --- PRC-CMV WTp53 143-p53 22,23-p53 consensus p53-binding site could be shown even allowing for two mismatches. Nevertheless, we tested for a p53 potential binding site for p53 in this 41 bp sequence by EMSA using a recombinant p53 polypeptide. The p53 cis b-actin element present in the p21 promoter was used as a positive control. As shown in Figure 6A, purified p53 can bind to Figure 3. Inhibition of APE1 promoter activity by WT but not mutant (/) p53. (A) p53 cells were transiently transfected with (0.5mg) of the p21 promoter (lane 2), but not to the APE1 promoter APE1 promoter–luciferase plasmid (4500/+65) and various amounts sequence (lanes 4 and 5), indicating a lack of direct p53 of WTp53 expression plasmid or control vector. Luciferase activity was binding to the APE1 promoter in vitro. measured at 48 h after transfection and normalized for the amount of protein. The mean  SD of five independent experiments performed in duplicate is shown. (B) Western analysis of the p53 level in the transfected with (0.5mg) of APE1 promoter–luciferase plasmid (/) transfected cell extracts used in (A). (C) p53 cells were cotrans- (4500/+65) and 0.05 mg of expression plasmid encoding WTp53 or fected with (0.5mg) of p21 promoter–luciferase plasmid and WTp53. mutant p53 (V143A) (22,23) or equivalent amount of empty vector. Luciferase activity was measured 48 h after transfection and normalized Other details are given above. (E) Western analysis of WT and mutant (/) for the amount of protein. (D) p53 cells were transiently p53 in cells lysates used in (D); P< 0.05; P< 0.001. Fold inhibition Fold inhibition Fold activation Nucleic Acids Research, 2008, Vol. 36, No. 5 1561 A 3000000 A (+1) (+65) SmaI −1779 Luciferase 4 APE1-LUC deletion constructs 500000 7 APE1-LUC (0.5mg) 4kb 1.8kb B 160 * * APE1-LUC construct 4kb 1.8kb 1 2 3 4 5 6 Figure 5. (A) Schematic diagram of the APE1 promoter 5 regulatory WTp53 (mg) -- 0.01 0.03 0.05 0.07 0.1 0.3 region showing the location of DNA fragments cloned upstream of the 0 0 luciferase-coding region. The nucleotides are numbered 5 () and 3 (+) Figure 4. Effect of WTp53 on APE1 promoter reporter activity. (/) from the transcription start site (+1). (B) p53 cells were (A) Basal promoter activity of APE1 promoter luciferase constructs cotransfected with expression plasmid for WTp53 (0.1mg) or empty containing 4500 or 1800 bp of APE1 promoter sequences linked to vector and individually for APE1 promoter–reporter plasmids (0.5mg) (/) the luciferase gene. (B) p53 cells were transiently transfected with 1–8, as indicated in (A). Luciferase activity was measured 48 h after 0.5mg of APE1 promoter–luciferase plasmid (1800/+65) and WTp53 transfection and normalized for the amount of protein. Fold inhibition or control vector as before. Luciferase activity was measured 48 h after was calculated as the ratio of luciferase activity from empty vector transfection as before; P< 0.05. transfected versus p53-transfected cells; the results represent the mean  SD in five independent experiments performed in duplicate; P< 0.001. Recruitment of p53 to the APE1 promoter is induced by cellular stress (/) Because p53 could exert its repressor activity via interac- chromatin fraction of p53 cells (Figure 6B). Similarly, tion with other transcription factors bound to their no significant enrichment of the APE1 promoter sequence cognate promoters, we used ChIP assay to test whether was observed in chromatin extracts from either cell line p53 is associated with the APE1 promoter in vivo (41). when non-specific IgG was used for immunoprecipitation (+/+) (/) (Figure 6B). An earlier study identified overlapping Sp1- p53 and p53 HCT116 cells were treated with CPT for 6 h, and subsequently with 1 mM disuccinimidyl binding sites within this APE1 promoter sequence (53). As glutarate, which produces protein–protein crosslinks, expected, significant enrichment of the APE1 promoter followed by further incubation with 1% formaldehyde to sequence was observed in the Sp1-specific immunocom- produce protein–DNA crosslinks. The chromatin frac- plexes from both cell lines (Figure 6B), indicating that Sp1 tions were isolated and fragmented by digestion. After is constitutively associated with the APE1 promoter in the (+/+) (/) immunoprecipitation with antibody to p53 or Sp1, ChIP chromatin fraction of both p53 and p53 cells. To assays were then performed as described in Materials and establish that p53 selectively binds to this (184 to Methods section. The amount of immunoprecipitated 143 bp) sequence, we used appropriate primer sequences APE1 promoter sequence was quantitated for each sample to amplify the region corresponding to 253 to 118 bp by PCR analysis with primers for amplification of the of the APE1 promoter. Figure 6C shows that this APE1 promoter region from 368 to +24 bp. Figure 6B sequence was selectively enriched in the p53 immuno- (+/+) (/) shows significant enrichment of this sequence in the complex from the p53 but not p53 HCT116 cells. immunocomplex with p53 antibody, indicating that p53 This confirms binding of p53 to this sequence in vivo. is associated with the APE1 promoter in vivo in HCT116 These results provide strong evidence that both p53 and (+/+) p53 cells. We performed appropriate control experi- Sp1 are associated with the 253 to 118 bp region in the ments to validate our results. Thus, no significant human APE1 promoter in vivo. enrichment of the APE1 promoter sequence was observed Although there is no evidence for a p53-specific cis by the PCR assay in the p53 immunoprecipitate from element in the APE1 promoter, the presence of p53 in the Fold inhibition Luciferase activity (R.L.U.) Fold Inhibition of APE1-LUC activity −467 −264 −184 −143 −118 1562 Nucleic Acids Research, 2008, Vol. 36, No. 5 p21 oligo. + + −− − − ++ Recombinant WTp53 − + − + APE1 oligo. − ++ 12 3 4 5 Antibodies Input IgG p53 Sp1 (+/+) HCT116 p53 (−/−) HCT116 p53 −368 Antibodies Input Ig Gp 53 Sp1 (+/+) HCT116 p53 (−/−) HCT116 p53 −253 −118 Figure 6. ChIP assay for in vivo association of p53 with APE1 promoter. (A) EMSA of purified p53 (60 ng, lane 4; 120 ng, lane 5) using P-labeled duplex oligo corresponding to the bases (184 to 142) from the APE1 promoter sequence or containing consensus p53-binding sequence from the (+/+) p21 promoter (lane 2) as a control. (B) p53 cells were treated with CPT for 9 h, the protein–DNA was crosslinked, and ChIP assays performed as described in Materials and Methods section. Immunoprecipitated APE1 promoter with the indicated antibody was amplified with primers as described in Materials and Methods section. Left panel, schematic diagram of the APE1 promoter showing the relative position of PCR primers used in the ChIP assays. APE1 promoter complex as indicated by our ChIP assay P53-induced APE1 repression is mediated by interference strongly supports the idea that p53 is recruited to the with Sp1 binding to the APE1 promoter APE1 promoter via binding to some other trans-acting Identification of one Sp1 site within the putative p53 factors. p53 has been shown to interact with several response element in the APE1 promoter raised the factors of the basal transcription machinery, e.g. TATA- possibility that p53 is recruited by Sp1 to the APE1 binding Protein (TBP) or the basal transcription factor promoter complex for inhibition (Figure 7B). ChIP Sp1, and also to recruit mSin3A/HDAC repressor analysis clearly demonstrated association of Sp1 and p53 complex to the promoter to inhibit transcription (41). in the APE1 promoter complex (Figure 6B). p53 has been Consistent with this, p53-mediated repression of several shown to bind Sp1 and interferes with its binding to the cis promoters has been shown to be reversed with the HDAC element for Sp1-mediated transcription (56–58). To test inhibitor, tricostatin A (TSA) (54,55). To test possible this possibility, we performed EMSA with APE1’s Sp1- involvement of HDAC in p53-mediated APE1 repression, binding sequence. Formation of specific protein–DNA we used the 1800/+65 APE1-Luc promoter–reporter complexes suggested the binding of Sp1 to the oligo plasmid which showed the highest promoter activity. At (/) (Figure 7C), which was competed with unlabeled oligo 24 h after cotransfection of HCT116 cells with the containing WT but not a mutated Sp1 oligo sequence. APE1 promoter–reporter and WTp53 plasmids, the cells These data confirm the specificity of Sp1 binding to this were treated with 100 ng/ml TSA. Figure 7A shows that sequence. Furthermore, a significant decrease in binding TSA reversed p53-mediated repression of luciferase to a significant extent, suggesting that the repression involves was observed with nuclear extract from p53-overexpres- (/) p53-dependent recruitment of HDAC to the APE1 sing HCT116 cells, suggesting that WTp53 interferes promoter. with Sp1 binding to the promoter. Nucleic Acids Research, 2008, Vol. 36, No. 5 1563 A We have shown that the levels of both APE1 mRNA and polypeptide were decreased after CPT treatment in (+/+) HCT116 p53 , but not in the isogenic HCT116 150 (/) (+/+) p53 cells. This effect on HCT116 p53 cells was specific, and not a consequence of general effects of genotoxic or other types of stress, because the same treatment did not affect APE1 expression in p53 null cells. 50 * This thus indicates direct relationship between the p53 status and APE1 downregulation. Reduced expression of −1800/+65 APE1-Luc(0.5µg) + + + + + + + + both endogenous APE1 and APE1 promoter-dependent WTp53 (µg) −− 0.05 0.05 0.1 0.1 0.3 0.3 luciferase activity by exogenous p53 in unstressed p53 null TSA (100 ng/ml) − + − + − + − + cells further confirmed that p53 downregulates APE1 −177 −143 expression. However, ectopic expression of WTp53 down- APE1 5′ AGA GGA GGG AGG CGA GGC TAA GCG TCT CCG TCA CGT 3′ regulated the episomal APE1 promoter to a much greater probe extent than the endogenous gene which was somewhat unexpected. It is possible that the APE1 promoter in the Transcriptional chromatinized plasmid is more sensitive to the effect of HindIII (−128) start site AFlII CCAAT (+1) (+65) p53 than the folded conformation in cellular chromatin. −264 Furthermore, the endogenous APE1 gene could be regulated by binding of transcription factors to cis elements absent in the episomal promoter. The V143A AP-1 Nrul p53 mutant lacking DNA-binding activity did not down- GRE Sp1 USF (−173) regulate the basal APE1 promoter as much as did WTp53, Nuclear extract − ++ + + which strongly suggests that p53’s DNA-binding ability APE1 oligo. ++++ + is a prerequisite for its repressor activity. However, the WTp53 (µg) −− − 0.6 0.6 APE1 oligo. − −− + + L22G, T23S double mutant with normal DNA-binding activity, but lacking trans-acting activity (52), was also unable to repress the APE1 promoter, which suggests that both DNA binding and transactivation abilities of p53 are required for the repression. Western analysis showing that p53 mutants were more stable than the WTp53 protein eliminated the possibility that the former’s inability to repress the APE1 promoter was due to reduced stability. Figure 7. Effect of TSA on p53-mediated repression of APE1 promoter Using functional analysis of nested deletion mutants, (/) activity. (A) p53 cells were transiently transfected with 0.5mg we mapped the basal APE1 promoter to a 143 bp of APE1 promoter–luciferase plasmid (1800/+65) and increasing amounts of expression plasmid encoding WTp53 or equivalent amounts segment 5 to the transcription start site. We also observed of empty vector. Twenty-four hours after transfection cells were treated that the candidate p53-responsive cis element(s) were with or without TSA (100 ng/ml) and luciferase activity was measured located in the 184 to 143 bp segment. However, 48 h after transfection and normalized for the amount of protein. The sequence analysis revealed no apparent p53-binding sites mean  SD of five independent experiments performed in duplicate is within this segment. Moreover, the recombinant p53 shown. (B) Sequence of the DNA probes for EMSA assay with APE1 promoter. Schematic diagram of the APE1 promoter showing some of polypeptide failed to bind to an oligo of this sequence the putative transcription factor binding sites (Sp1; USF; glucocorticoid (184 to 143). At the same time we have provided direct response element (GRE); activator protein 1 (AP-1); and CCAAT box- evidence from ChIP assays that p53 was bound to the (/) like sequence). (C) EMSA with nuclear extracts of p53 cells 253 to 118 bp segment sequence in vivo. Together, these transfected with empty vector or WTp53 expression plasmid using P-labeled duplex oligo corresponding to bases 177 to 143 from data suggest that APE1 repression by p53 is not mediated APE1 promoter; P< 0.05. via direct cis element binding, but rather involves p53’s indirect recruitment to the promoter by other transcrip- tion factors. p53 has been shown to repress many other DISCUSSION promoters that lack p53-specific cis elements, indicating APE1 is often overexpressed in tumor cells compared to that the mechanisms of p53-mediated repression are its basal level in many untransformed cells lines. varied and complex (41,58,60,61). Such mechanisms Downregulation of APE1 was found to sensitize tumor could include (i) interference with transcriptional activa- cells to chemotherapy with induction of apoptosis tors, (ii) interference with the basal transcription machin- (26,27,29). Thus, elucidating the molecular basis for ery and (iii) compaction of the chromatin structure at the variable APE1 expression is important from both basic promoter sites by recruitment of HDACs and other and clinical perspectives. Although several studies have histone-modifying enzymes (41). shown that p53 participates in DNA repair processes, and The exact mechanism by which p53 represses the APE1 stimulates BER via its direct interaction with APE1 and expression is not clear. We have shown that candidate DNA polymerase b, whether p53 regulates APE1 level p53-responsive element(s) were located in the 184 to itself was not shown before (59). This report provides the 143 bp segment that includes one Sp1 site. An earlier first evidence that p53 downregulates APE1 expression. study using in vitro DNase I footprinting analysis with Fold inhibition 1564 Nucleic Acids Research, 2008, Vol. 36, No. 5 purified Sp1 polypeptide showed a distinct footprint CA98664 to T.I.), American Heart Association grant spanning 169 to 148 bp in the APE1 promoter (53). (AHA#0565008Y to K.B.). Amira Zaky was supported by Consistent with this, our ChIP assay showed constitutive a Government of Egypt scholarship for research at (+/+) Sp1 binding within this region in both p53 and University of Texas medical Branch. Funding to pay the (/) p53 cells, indicating that the Sp1 site is functional Open Access publication charges were waived by Oxford in vivo. Moreover, we observed that Sp1 remains bound to University Press. the APE1 promoter after p53 activation by CPT, Conflict of interest statement. None declared. suggesting that both p53 and Sp1 can simultaneously occupy the same promoter region. 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Regulation of the human AP-endonuclease (APE1/Ref-1) expression by the tumor suppressor p53 in response to DNA damage

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Abstract

Published online 21 January 2008 Nucleic Acids Research, 2008, Vol. 36, No. 5 1555–1566 doi:10.1093/nar/gkm1173 Regulation of the human AP-endonuclease (APE1/Ref-1) expression by the tumor suppressor p53 in response to DNA damage 1,2 3 3 1 Amira Zaky , Carlos Busso , Tadahide Izumi , Ranajoy Chattopadhyay , 2 1 1, Ahmad Bassiouny , Sankar Mitra and Kishor K. Bhakat * Department of Biochemistry and Molecular Biology, Sealy Center for Molecular Medicine, University of Texas Medical Branch, TX-77555, Galveston, USA, Department of Biochemistry, Alexandria University, Alexandria, Egypt, 21511 and Department of Otolaryngology, Louisiana State University Health Science Center, LA 70112, New Orleans, USA Received October 31, 2007; Revised December 19, 2007; Accepted December 20, 2007 role in the base excision repair (BER) pathway for ABSTRACT damaged bases induced by reactive oxygen species and The human AP-endonuclease (APE1/Ref-1), an alkylating agents, and for abasic (AP) sites generated essential multifunctional protein, plays a central after excision of oxidized and alkylated bases by DNA role in the repair of oxidative base damage via glycosylases (1–3). As an endonuclease, APE1 cleaves the 0 0 the DNA base excision repair (BER) pathway. The AP site to generate 3 -OH and 5 -deoxyribose phosphate mammalian AP-endonuclease (APE1) overexpres- termini, and the 3 -OH terminus is utilized by a DNA sion is often observed in tumor cells, and confers polymerase, usually DNA polymerase b, for repair synthesis in mammalian cells (4). APE1 was independently resistance to various anticancer drugs; its down- identified as a reductive activator of the c-Jun transcrip- regulation sensitizes tumor cells to those agents tion factor in vitro, and named Ref-1 (5). Subsequently, via induction of apoptosis. Here we show that wild several other transcription factors (including p53, type (WT) but not mutant p53 negatively regulates hypoxia-inducible factor and NF-kB) were also found to APE1 expression. Time-dependent decrease was be activated by APE1 (6–8). In addition, APE1 directly observed in APE1 mRNA and protein levels in the 2+ acts as a trans-acting factor by binding to negative Ca (+/+) human colorectal cancer line HCT116 p53 , but 2+ response elements (nCaRE) during Ca -dependent not in the isogenic p53 null mutant after treatment repression of the human parathyroid hormone and renin with camptothecin, a DNA topoisomerase I inhibitor. genes (9–11). Furthermore, ectopic expression of WTp53 in the Given its multiple functions, it is not surprising that p53 null cells significantly reduced both endogen- APE1 expression is regulated in vivo in a complex manner. ous APE1 and APE1 promoter-dependent luciferase We were one of the first to show the activation of APE1 gene by oxidative stress; several other laboratories sub- expression in a dose-dependent fashion. Chromatin sequently confirmed our observation (12–14). Moreover, immunoprecipitation assays revealed that endogen- based on the identification of the nCaREs in the APE1 ous p53 is bound to the APE1 promoter region that promoter itself, we suggested that APE1 regulates its own includes a Sp1 site. We show here that WTp53 expression (15). While cell cycle-dependent expression of interferes with Sp1 binding to the APE1 promoter, APE1 was observed in NIH3T3 cells (16), the basal which provides a mechanism for the downregulation expression of APE1 is variable. The increased APE1 level of APE1. Taken together, our results demonstrate is often observed in tumor cells including prostate, that WTp53 is a negative regulator of APE1 expres- osteosarcomas, lung and cervical carcinomas compared sion, so that repression of APE1 by p53 could to normal tissues (17–20). In other types of cancer, provide an additional pathway for p53-dependent subcellular localization of APE1 is altered relative to induction of apoptosis in response to DNA damage. that in normal tissues (21–23). Tumor cells often over- express APE1 and are resistant to chemotherapeutic drugs and ionizing radiation (19,24,25). In contrast, INTRODUCTION siRNA-mediated downregulation of APE1 induced apop- The mammalian AP-endonuclease (APE1) is a ubiquitous tosis of many tumor cell types and enhanced cell and remarkably multifunctional protein. It plays a central sensitization to ionizing radiation and chemotherapeutic *To whom correspondence should be addressed. Tel: 409 772 1779; Fax: 409 747 8608; Email: [email protected] 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 1556 Nucleic Acids Research, 2008, Vol. 36, No. 5 agents (26–29). We recently showed that APE1 inactiva- MATERIALS AND METHODS tion-induced apoptosis in mouse embryo fibroblasts Cell culture and treatment (MEF) conditionally nullizygous for endogenous APE1, The human colorectal adenocarcinoma lines, HCT116 an effect that could be prevented by ectopic expression of (+/+) (/) p53 with WTp53 and p53 null HCT116 p53 human APE1 (29). Using a complementation assay, we (a gift from Dr B. Vogelstein, Johns Hopkins University also showed that both the repair and transcription School of Medicine) were grown in McCoy’s 5A (Gibco regulatory functions of APE1 are required to prevent Life Technologies, Carlsbad, CA, USA) medium supple- apoptosis of MEF (29). Thus, elucidating the molecular mented with 10% fetal bovine serum (Sigma-Aldrich, mechanisms controlling APE1 expression has profound St Louis, MO, USA), 100 U/ml penicillin and 100mg/ml implications for cancer therapy from both basic and streptomycin (Gibco BRL, Carlsbad, CA, USA) in a 5% clinical perspectives. C0 incubator at 378C. Cells were treated with 200 nM The tumor suppressor gene p53, dubbed ‘the guardian camptothecin (CPT) (Sigma, St Louis, MO, USA) and/or of the genome’, encodes a sequence-specific transcription 30mM pifithrin-a (PFT-a) (Sigma, St Louis, MO, USA). factor that activates a variety of cellular genes in response to DNA damage, hypoxia and other stress signals (30,31). Plasmids and luciferase assays p53 is one of the most commonly mutated genes in human cancers; 50% of all human cancers lack the wild type Generation of APE1 promoter–reporter plasmids contain- (WT) p53 gene allele, and these tumors respond poorly ing DNA fragments (4800/+65) and (1800/+65) of to chemotherapy (32,33). In response to DNA damage or the 5 regulatory region was described earlier (15). Other other cellular stresses, p53 is activated and induces cell reporter plasmids with various lengths of the APE1 5 regulatory region were generated by PCR and cloned into cycle arrest or apoptosis, depending on the severity of the the luciferase reporter vector pGL3 (Promega, Madison damage and the context of the cell cycle, and thus helps (/) WI, USA) using standard procedures. HCT116 p53 to maintain genomic stability and prevents cancer (34). cells were cotransfected with 500 ng of the APE1-reporter Once activated, p53 has the ability to arrest cell cycle by plasmids and expression plasmids for WT or mutant transactivation of Waf-1/p21, 14-3-3d etc., or induce cells [Val143Ala (V143A) and L22G, T23S] p53 or equivalent to undergo apoptosis by both transcription-dependent and amounts of empty vector using LipofectAMINE 2000 -independent mechanisms (35–37). p53 activates many (Invitrogen, Life Technologies, Carlsbad, CA, USA), downstream target genes involved in the mitochondrial according to the manufacturer’s instructions. At 48 h signaling pathway during apoptosis including BAX, after the transfections, cells were lysed with the reporter PUMA and the death receptor (38–40). In addition, p53 lysis buffer (Promega), and the luciferase activity in the has been shown to downregulate many genes, including cell lysates was measured in a luminometer (AutoLumant cell survival genes such as survivin, bcl-2, IGF1R and DNA LB593, Berthold, Oak Ridge, TN, USA) by the luciferase repair gene O -methylguanine-DNA-methyltranferase assay kit (Promega, Madison, WI, USA) according to the (MGMT) with the net result of facilitating apoptosis manufacturer’s protocol. The luciferase activity was induction (41–46). normalized to the amount of protein in the lysate. Potential link between APE1 expression and p53 status in various tumors and their resistance to chemotherapeu- Preparation of total cell extract and western blot analysis tic drugs have been documented (24,47). Despite several studies showing the involvement of APE1 and p53 in drug HCT116 cells were lysed in a lysis buffer (50 mM Tris- resistance induction in tumor cells, the question of HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% TritonX- whether p53 regulates APE1 expression after genotoxic 100, and protease inhibitor cocktail (Roche, Nutley NJ, stress has not been addressed. Elucidating the direct role USA) as described earlier (48). Whole-cell extracts (25 mg) of p53 on APE1 gene expression in human cells should be were subjected to 12.5% SDS–PAGE and transferred to a of major clinical significance, and a clear understanding of nitrocellulose membrane (Trans-Blot 0.2mm, Bio-Rad, Hercules, CA, USA). Western immunoblotting analyses the mechanisms by which p53 controls APE1 expression with mouse monoclonal antip53 antibody (sc-DO-1, could help in effective use of chemotherapeutic agents in dilution 1:200, Santa Cruz Biotechnology, CA, USA), the treatment of tumors expressing WT or mutant p53. rabbit polyclonal antiAPE1 (dilution 1:2000), or rabbit In this study, we show that activation of p53 after geno- monoclonal antib-actin antibody (Sigma, 1:1000 dilution) toxic stress downregulates APE1 expression in HCT116 (+/+) were carried out using enhanced chemiluminescence assay p53 human colon carcinoma cells, but not in the p53 (ECL kit, Amersham, London, UK). null mutants derived there from. Transgenic expression of WTp53 in p53 null cells downregulate both endogenous RNA isolation and RT-PCR analysis APE1 level and expression of a reporter whose activity is dependent on APE1 promoter activity. We also present Total RNA was extracted using Rneasy Mini kit evidence that p53 is associated with the endogenous APE1 (QIAGEN) according to the manufacturer’s protocol. promoter in vivo, and that interference of specificity For quantitative, real-time RT-PCR, one step RT-PCR protein (Sp1) binding to the APE1 promoter by WTp53 was performed with 100 ng of cellular RNA for both the and subsequent recruitment of histone deacetylase target gene and an endogenous control in singleplex tubes (HDAC) to the promoter could explain downregulation using TaqMan one-step RT-PCR master mixture reagent of APE1 by WTp53. kit (P/N 4309196). TaqMan MGB probes (FAMTM Nucleic Acids Research, 2008, Vol. 36, No. 5 1557 dye-labeled) for APEX1 gene and 18s rRNA (VICTM-dye Dynamics, Piscataway, NJ, USA) and analyzed using labeled probe) TaqMan assay reagent (P/N 4319413E) IMAGEQUANT software. for endogenous control (assay-on-DemandTM (P/N 4331182) were used. The cycling parameters in an ABI 7000 (Applied Biosystem, Foster City, CA, USA) thermal cycler were as follows: reverse transcription at 488C for RESULTS 30 min. AmpliTaq activation 958C for 10 min, denatura- WTp53 downregulates APE1 mRNA and protein levels tion 958C for 15s, and annealing/extension 608C for 1 min. We examined the effect of WTp53 on APE1 expression The amount of target was calculated after normalization (+/+) (/) in HCT116 p53 and HCT116 p53 cell lines. To to the 18s RNA as an endogenous control. activate p53, these cells were treated with CPT, a DNA Chromatin immunoprecipitation (ChIP) assay topoisomerase I inhibitor, and total RNA was isolated (+/+) (/) at the indicated times for quantitation of APE1 mRNA HCT116 p53 and HCT116 p53 cells were treated levels by real-time RT-PCR. We observed a significant with CPT at final concentration of 200 nM for 6 h unless decrease in the APE1 mRNA level at 18 h after treatment otherwise indicated. Cells were washed twice with ice-cold with CPT with simultaneous activation of WTp53 in PBS, fixed with 1% formaldehyde for 10 min at RT, (+/+) HCT116 p53 cells (Figure 1A). Treatment with CPT washed twice with cold PBS, and harvested in cell scraping also reduced APE1 mRNA level in p53-negative HCT116 solution (1X PBS and 0.5 mM PMSF). Pellets were (/) p53 cells, although not to the same extent as in the collected by centrifugation at 1200 rpm for 10 min at 48C (+/+) p53 cells indicating a relationship between the p53 and resuspended in ice-cold lysis buffer (Active Motif, status and the downregulation of APE1 expression. ChIP-IT, Carlsbad, CA, USA, supplemented with 0.5 mM Treatment with CPT also induced the p21 level in PMSF and 0.5 mM protease inhibitor cocktail), and then (+/+) HCT116 p53 cells concomitant with enhanced p53 incubated on ice for 30 min. The DNA fragments were levels (Figure 1B). Activation of the p21 gene was used to digested with mung bean nuclease to an average size of monitor in vivo function of p53. Although a transient 200–500 bp, as empirically estimated by agarose gel increase in the APE1 protein level was observed soon after electrophoresis of the digest. Immunoprecipitations were (1 h) CPT treatment, consistent with the decreased APE1 performed with antip53 (DO-1), IgG (as a negative mRNA level, the APE1 polypeptide level was also control) and antiSp1 (as a positive control) antibodies. (+/+) significantly reduced in HCT116 p53 cells at 18 h After sequential washing, DNA–protein complexes were after treatment with CPT (Figure 1B, right panel). eluted with elution buffer (1% SDS, 0.1 M NaHCO , We further confirmed the role of p53 by treating 0.01 mg/ml herring sperm DNA). The crosslinks were cells with the water soluble p53 inhibitor PFT-a, which reversed by heating at 658C overnight, and treatment with blocked activation of p53-regulated genes, including proteinase K (0.17mg/ml) for 1 h, and the DNA then cyclin G, p21/Waf-1 and mdm2, and also inhibits isolated using a column (Active Motif, ChIP-IT) accord- apoptosis (49–51). Treatment with PFT-a attenuated ing to the manufacturer’s guidelines. Recovered DNA was (+/+) APE1 repression after CPT treatment in p53 cells suspended in 50ml of TE. PCR amplification of DNA was carried out with diluted aliquots using appropriate (Figure 1C), while it had no effect on the APE1 level in (/) primers to generate PCR products spanning +24 to p53 cells (Figure 1D). Taken together, these results 368 bp and 118 to 253 bp of the APE1 promoter suggest that downregulation of APE1 expression is region. The PCR products were separated by 1.5% p53-dependent. agarose electrophoresis in Tris-borate-EDTA buffer and To further confirm that WTp53 acts as a negative stained with ethidium bromide. regulator of APE1 expression, we investigated the effect of WTp53 overexpression on the APE1 protein and mRNA Electrophoretic mobility shift assay (EMSA) levels in HCT116 p53 null cells. Various amounts of expression plasmid for WTp53 were used for transfection Electrophoretic mobility shift analyses were performed of p53 null cells, and the expression of p53 and p21 was as described earlier (38) with some modifications. The 0 32 0 confirmed by western analysis of the cell extracts 5 P-labeled oligo 5 -AGAGAGGGAGGCGAGGCTA (Figure 2A). Overexpression of p53 decreased APE1 AGCGTCTCCGTCACGT-3 (potential p53-binding site mRNA and protein levels in a dose-dependent manner on APE1 promoter) and 5 -TTG AAC ATG TCC CAA (Figure 2A and B). To confirm that ectopic p53 was CAT GTT GA-3 containing the p53 consensus sequence transcriptionally active, we measured the p21 levels in from the human p21 promoter were annealed with nuclear extracts of p53-transfected cells (Figure 2C), and appropriate complementary stands to generate duplex its binding to double-stranded oligonucleotides containing oligos. The DNA (50 fmol) was then incubated with p53 (/) the p53-binding motif of the p21 promoter. Figure 2C or nuclear extracts (5mg) from HCT116 p53 cells for shows predominant localization of p53 in nuclear fraction 20 min at 258C in a buffer containing 40 mM HEPES- of cells after ectopic expression of WTp53. EMSA of the KOH, pH 7.5, 50 mM KCl, 1 mM MgCl , 0.5 mM EDTA, same extract confirmed the presence of WTp53 which 0.5 mM DTT, 10% glycerol, 1 mg of poly dI-dC (Sigma). formed a shifted complex with the p53 cis sequence After electrophoresis in non-denaturing 5% polyacryla- mide gels in Tris-borate buffer at 48C, the gels were (Figure 2D). Together, these results further confirm that dried and exposed to PhosphorImager (Molecular p53 acts as a repressor of APE1 expression. 1558 Nucleic Acids Research, 2008, Vol. 36, No. 5 1.4 1.2 HCT116 p53 (+/+) HCT116 p53 (−/−) 0.8 0.6 0.4 0.2 c 1h3h6h 18h Time (h) 0 1 3 6 18 24 p53 APE1 p21 b-actin CPT (200 nM) −−++ PFT-a (30 µM) −− ++ p53 APE1 p21 b-actin CPT (200 nM) − + − + p53 PFT-(30 µM) −− ++ APE1 b-actin (+/+) Figure 1. Repression of APE1 gene expression in HCT116 p53 cells after CPT treatment. (A) Real-time RT-PCR analysis of APE1 mRNA (+/+) (/) from HCT116 p53 and HCT116 p53 cells at indicated time points after CPT treatment. Results correspond to mean  SD from three (+/+) separate experiments. (B) After treating HCT116 p53 cells with CPT (200 nM), as described under Materials and Methods section, total lysates of cells harvested at the indicated times were analyzed for p53, APE1 and p21 levels by western blotting. b-actin was used as the loading control. Right panel, graphical representation of APE1 protein level (normalized to b-actin) at indicated time points after CPT treatment. Results correspond (+/+) (/) to mean  SD from three separate experiments. (C and D) HCT116 p53 or p53 cells were treated with CPT (200 nM) for 24 h in the presence or absence of the p53 inhibitor PFT-a and total lysates were analyzed for p53, APE1 and p21 levels by western blotting. b-actin was used as the loading control. Right panel, graphical representation of APE1 protein level (normalized to b-actin) after treatment with CPT or PFT-a. Results correspond to mean  SD from three separate experiments; P< 0.05. APE1 promoter activity is repressed by WTp53, but not dose-dependent decrease in APE1 promoter activity (up to by mutant p53 250-fold compared to the vector control, Figure 3A). Increase in p53 expression was confirmed by western To determine whether p53-mediated downregulation analysis of the same cells extract with antip53 antibody of APE1 occurred at the promoter level, we exam- (Figure 3B). Because p53 is an activator of the p21 gene, ined the effect of p53 overexpression on APE1 promo- we simultaneously examined the effect of ectopic expres- ter-dependent luciferase activity in a transient reporter sion of p53 on p21 promoter-dependent luciferase activity. expression assay. We cotransfected an APE1 promoter luciferase reporter construct (4000/+65 APE1-Luc, The increase in p21 promoter-dependent luciferase activity containing the APE1 promoter sequence from 4000 to with increasing amounts of input p53 plasmid indicated +65 bp) and the WTp53 expression plasmid into HCT116 that the inhibitory effect of p53 on APE1 promoter was (/) p53 cells. Ectopic expression of WTp53 induced a not due to general inhibition of transcription (Figure 3C). (+/+) HCT116 p53 (−/−) (+/+) HCT116 p53 HCT116 p53 APE1 mRNA level (normalized to 18s) Nucleic Acids Research, 2008, Vol. 36, No. 5 1559 WTp53 1.2 plasmid (mg) mock 0.3 0.6 1.2 p53 0.8 0.6 APE1 0.4 0.2 p21 b-actin mock 0.3 0.6 1.2 WTp53 plasmid (mg) B 2 1.8 (−) PFT-a 1.6 1.4 (+) PFT-a 1.2 0.8 0.6 * 0.4 * 0.2 WTp53 plasmid (mg) --- 0.3 0.6 1.2 CD (−/−) p53 nuclear extract − ++++++ Empty vector − −− 0.6 −− 0.6 WTp53 WTp53 plasmid 0.3 0.6 0.6 plasmid (mg) − −−− mock 0.3 0.6 Anti-p53 antibody − − −−− + + p53 Lamin B p21 Figure 2. Decreased APE1 protein and mRNA levels after ectopic expression of WTp53 in p53 null cells. (A) Total cell extracts were analyzed for p53, APE1 and p21 levels by western blotting. b-actin was used as the loading control. Right panel, graphical representation of APE1 protein level (normalized to b-actin) after transfection with various amounts of WTp53 expression plasmid. Results correspond to mean  SD from three separate (/) experiments. (B) Real-time RT-PCR analysis of APE1 mRNA levels in HCT116 p53 cells transfected with various amounts of WTp53 expression plasmid and incubated with PFT-a or vehicle for 48 h. Results correspond to mean  SD from three separate experiments. (C) Western analysis of p53 and p21 levels in nuclear fraction in p53 null cells after ectopic expression of WTp53. Lamin B was used for nuclear extracts loading control. (D) EMSA of nuclear extracts used in (C) using P-labeled duplex oligo from p21 promoter containing consensus p53-binding sequence; P< 0.05. (/) Moreover, we observed that CMV promoter-dependent effect (Figure 3D). Western analysis of p53 cells b-galactosidase and thymidine kinase promoter-dependent lysates transiently transfected with p53 expression plas- renilla-luciferase (pRL-TK, Promega) levels were also mids revealed that the p53 mutant polypeptides were more stable than the WT protein, and were present at higher increased due to overexpression of WTp53 for unknown levels (2- to 3-fold, Figure 3E). These results indicate that reasons (data not shown). We thus could not therefore use despite these higher levels, mutant p53 could not inhibit these reporter plasmids for normalizing transfection the APE1 promoter activity. efficiency. In any case, our results indicate that the p53- dependent repression is APE1 promoter specific, and Identification of p53-responsive cis element(s) not a general phenomenon. We also examined the effects in APE1 promoter of two p53 point mutants on APE1 promoter activity namely, V143A, a missense mutation which inactivates To identify the cis element(s) responsible for p53-mediated p53’s sequence-specific DNA binding, and a Leu22Gln downregulation of the APE1 promoter, we carried out Trp23Ser (22,23), double mutant which retains the ability promoter deletion analysis. We have shown earlier that to bind p53-cis sequences but lacks transcriptional the APE1 promoter contains multiple negative regulatory activation ability (52). Overexpression of WTp53 signifi- and enhancer elements, including two nCaRE-B elements cantly decreased luciferase activity under the control of upstream (4000 to 1800 bp) of the basal promoter (15). the APE1 promoter (reaching up to 60-fold with 50 ng of To test whether p53-induced repression of APE1 promoter WTp53), while the mutants had no significant inhibitory activity is mediated through the cis elements located APE1 mRNA level (normalized to 18s) Fold change in APE1 protein level 1560 Nucleic Acids Research, 2008, Vol. 36, No. 5 (/) A in this region, we cotransfected HCT116 p53 cells with WTp53 and the reporter plasmid containing 1800 to +65 bp (1800/+65 APE1-Luc) of the APE1 promo- ter. Deletion of the promoter sequence from 4000 to 1800 bp significantly increased the basal promoter activity (9-fold, Figure 4A), confirming our earlier observation about the presence of negative regulatory elements within this sequence (15). Furthermore, ectopic expression of WTp53 caused a decrease in the promoter (1800/+65 APE1-Luc) activity in a dose-dependent −4000/+65 APE1-Luc (0.5µg)++ + + + + + + WTp53 (mg) -- 0.01 0.03 0.05 0.07 0.1 0.3 0.5 manner (Figure 4B). We then carried out detailed mapping of the p53 regulatory sequence by using a series of 5 promoter p53 B deletion constructs. DNA fragments of the 5 regulatory region (shown in Figure 5A) were cloned upstream of the b-actin luciferase coding region and their promoter activity was determined by cotransfection as before. All deletion constructs except 7 and 8 showed basal promoter activity 3.5 that was abolished by deleting residues 143 to 118 (Figure 5A and B) indicating that the sequence upstream of 118 is required for basal promoter activity. Although 2.5 deletion of 4000 to 1800 decreased p53-mediated downregulation of promoter activity by 1.5-fold, lucifer- 1.5 ase activity of all the deletion constructs was significantly downregulated after cotransfection with WTp53 (Figure 5B). However, elimination of the sequence from 0.5 184 to 143 (construct number 6, Figure 5A) abolished the inhibitory effect of p53 (Figure 5B), indicating that the cis element involved in p53-dependent repression is localized within this sequence. Furthermore, deletion of WTp53 (ng) --- 10 50 the +65 bp region downstream to the transcription start site (construct number 5, Figure 5A) had no effect on p53- mediated repression, indicating that these sequences are * * not required for repression (Figure 5B). Thus the p53- APE1-Luc responsive cis elements appeared to be located within PRC-cmv WTp53 the proximal region (184 to 143 bp) of the APE1 143-p53 22.23-p53 promoter. Lack of direct binding of p53 to the APE1 promoter in vitro To identify the putative p53-responsive cis element(s) in the 41 bp (184 to 143) APE1 promoter sequence, we performed a computer search for potential transcription factor binding sites. Putative binding sites were identified one each for Sp1 and upstream factor (USF); however, no --- PRC-CMV WTp53 143-p53 22,23-p53 consensus p53-binding site could be shown even allowing for two mismatches. Nevertheless, we tested for a p53 potential binding site for p53 in this 41 bp sequence by EMSA using a recombinant p53 polypeptide. The p53 cis b-actin element present in the p21 promoter was used as a positive control. As shown in Figure 6A, purified p53 can bind to Figure 3. Inhibition of APE1 promoter activity by WT but not mutant (/) p53. (A) p53 cells were transiently transfected with (0.5mg) of the p21 promoter (lane 2), but not to the APE1 promoter APE1 promoter–luciferase plasmid (4500/+65) and various amounts sequence (lanes 4 and 5), indicating a lack of direct p53 of WTp53 expression plasmid or control vector. Luciferase activity was binding to the APE1 promoter in vitro. measured at 48 h after transfection and normalized for the amount of protein. The mean  SD of five independent experiments performed in duplicate is shown. (B) Western analysis of the p53 level in the transfected with (0.5mg) of APE1 promoter–luciferase plasmid (/) transfected cell extracts used in (A). (C) p53 cells were cotrans- (4500/+65) and 0.05 mg of expression plasmid encoding WTp53 or fected with (0.5mg) of p21 promoter–luciferase plasmid and WTp53. mutant p53 (V143A) (22,23) or equivalent amount of empty vector. Luciferase activity was measured 48 h after transfection and normalized Other details are given above. (E) Western analysis of WT and mutant (/) for the amount of protein. (D) p53 cells were transiently p53 in cells lysates used in (D); P< 0.05; P< 0.001. Fold inhibition Fold inhibition Fold activation Nucleic Acids Research, 2008, Vol. 36, No. 5 1561 A 3000000 A (+1) (+65) SmaI −1779 Luciferase 4 APE1-LUC deletion constructs 500000 7 APE1-LUC (0.5mg) 4kb 1.8kb B 160 * * APE1-LUC construct 4kb 1.8kb 1 2 3 4 5 6 Figure 5. (A) Schematic diagram of the APE1 promoter 5 regulatory WTp53 (mg) -- 0.01 0.03 0.05 0.07 0.1 0.3 region showing the location of DNA fragments cloned upstream of the 0 0 luciferase-coding region. The nucleotides are numbered 5 () and 3 (+) Figure 4. Effect of WTp53 on APE1 promoter reporter activity. (/) from the transcription start site (+1). (B) p53 cells were (A) Basal promoter activity of APE1 promoter luciferase constructs cotransfected with expression plasmid for WTp53 (0.1mg) or empty containing 4500 or 1800 bp of APE1 promoter sequences linked to vector and individually for APE1 promoter–reporter plasmids (0.5mg) (/) the luciferase gene. (B) p53 cells were transiently transfected with 1–8, as indicated in (A). Luciferase activity was measured 48 h after 0.5mg of APE1 promoter–luciferase plasmid (1800/+65) and WTp53 transfection and normalized for the amount of protein. Fold inhibition or control vector as before. Luciferase activity was measured 48 h after was calculated as the ratio of luciferase activity from empty vector transfection as before; P< 0.05. transfected versus p53-transfected cells; the results represent the mean  SD in five independent experiments performed in duplicate; P< 0.001. Recruitment of p53 to the APE1 promoter is induced by cellular stress (/) Because p53 could exert its repressor activity via interac- chromatin fraction of p53 cells (Figure 6B). Similarly, tion with other transcription factors bound to their no significant enrichment of the APE1 promoter sequence cognate promoters, we used ChIP assay to test whether was observed in chromatin extracts from either cell line p53 is associated with the APE1 promoter in vivo (41). when non-specific IgG was used for immunoprecipitation (+/+) (/) (Figure 6B). An earlier study identified overlapping Sp1- p53 and p53 HCT116 cells were treated with CPT for 6 h, and subsequently with 1 mM disuccinimidyl binding sites within this APE1 promoter sequence (53). As glutarate, which produces protein–protein crosslinks, expected, significant enrichment of the APE1 promoter followed by further incubation with 1% formaldehyde to sequence was observed in the Sp1-specific immunocom- produce protein–DNA crosslinks. The chromatin frac- plexes from both cell lines (Figure 6B), indicating that Sp1 tions were isolated and fragmented by digestion. After is constitutively associated with the APE1 promoter in the (+/+) (/) immunoprecipitation with antibody to p53 or Sp1, ChIP chromatin fraction of both p53 and p53 cells. To assays were then performed as described in Materials and establish that p53 selectively binds to this (184 to Methods section. The amount of immunoprecipitated 143 bp) sequence, we used appropriate primer sequences APE1 promoter sequence was quantitated for each sample to amplify the region corresponding to 253 to 118 bp by PCR analysis with primers for amplification of the of the APE1 promoter. Figure 6C shows that this APE1 promoter region from 368 to +24 bp. Figure 6B sequence was selectively enriched in the p53 immuno- (+/+) (/) shows significant enrichment of this sequence in the complex from the p53 but not p53 HCT116 cells. immunocomplex with p53 antibody, indicating that p53 This confirms binding of p53 to this sequence in vivo. is associated with the APE1 promoter in vivo in HCT116 These results provide strong evidence that both p53 and (+/+) p53 cells. We performed appropriate control experi- Sp1 are associated with the 253 to 118 bp region in the ments to validate our results. Thus, no significant human APE1 promoter in vivo. enrichment of the APE1 promoter sequence was observed Although there is no evidence for a p53-specific cis by the PCR assay in the p53 immunoprecipitate from element in the APE1 promoter, the presence of p53 in the Fold inhibition Luciferase activity (R.L.U.) Fold Inhibition of APE1-LUC activity −467 −264 −184 −143 −118 1562 Nucleic Acids Research, 2008, Vol. 36, No. 5 p21 oligo. + + −− − − ++ Recombinant WTp53 − + − + APE1 oligo. − ++ 12 3 4 5 Antibodies Input IgG p53 Sp1 (+/+) HCT116 p53 (−/−) HCT116 p53 −368 Antibodies Input Ig Gp 53 Sp1 (+/+) HCT116 p53 (−/−) HCT116 p53 −253 −118 Figure 6. ChIP assay for in vivo association of p53 with APE1 promoter. (A) EMSA of purified p53 (60 ng, lane 4; 120 ng, lane 5) using P-labeled duplex oligo corresponding to the bases (184 to 142) from the APE1 promoter sequence or containing consensus p53-binding sequence from the (+/+) p21 promoter (lane 2) as a control. (B) p53 cells were treated with CPT for 9 h, the protein–DNA was crosslinked, and ChIP assays performed as described in Materials and Methods section. Immunoprecipitated APE1 promoter with the indicated antibody was amplified with primers as described in Materials and Methods section. Left panel, schematic diagram of the APE1 promoter showing the relative position of PCR primers used in the ChIP assays. APE1 promoter complex as indicated by our ChIP assay P53-induced APE1 repression is mediated by interference strongly supports the idea that p53 is recruited to the with Sp1 binding to the APE1 promoter APE1 promoter via binding to some other trans-acting Identification of one Sp1 site within the putative p53 factors. p53 has been shown to interact with several response element in the APE1 promoter raised the factors of the basal transcription machinery, e.g. TATA- possibility that p53 is recruited by Sp1 to the APE1 binding Protein (TBP) or the basal transcription factor promoter complex for inhibition (Figure 7B). ChIP Sp1, and also to recruit mSin3A/HDAC repressor analysis clearly demonstrated association of Sp1 and p53 complex to the promoter to inhibit transcription (41). in the APE1 promoter complex (Figure 6B). p53 has been Consistent with this, p53-mediated repression of several shown to bind Sp1 and interferes with its binding to the cis promoters has been shown to be reversed with the HDAC element for Sp1-mediated transcription (56–58). To test inhibitor, tricostatin A (TSA) (54,55). To test possible this possibility, we performed EMSA with APE1’s Sp1- involvement of HDAC in p53-mediated APE1 repression, binding sequence. Formation of specific protein–DNA we used the 1800/+65 APE1-Luc promoter–reporter complexes suggested the binding of Sp1 to the oligo plasmid which showed the highest promoter activity. At (/) (Figure 7C), which was competed with unlabeled oligo 24 h after cotransfection of HCT116 cells with the containing WT but not a mutated Sp1 oligo sequence. APE1 promoter–reporter and WTp53 plasmids, the cells These data confirm the specificity of Sp1 binding to this were treated with 100 ng/ml TSA. Figure 7A shows that sequence. Furthermore, a significant decrease in binding TSA reversed p53-mediated repression of luciferase to a significant extent, suggesting that the repression involves was observed with nuclear extract from p53-overexpres- (/) p53-dependent recruitment of HDAC to the APE1 sing HCT116 cells, suggesting that WTp53 interferes promoter. with Sp1 binding to the promoter. Nucleic Acids Research, 2008, Vol. 36, No. 5 1563 A We have shown that the levels of both APE1 mRNA and polypeptide were decreased after CPT treatment in (+/+) HCT116 p53 , but not in the isogenic HCT116 150 (/) (+/+) p53 cells. This effect on HCT116 p53 cells was specific, and not a consequence of general effects of genotoxic or other types of stress, because the same treatment did not affect APE1 expression in p53 null cells. 50 * This thus indicates direct relationship between the p53 status and APE1 downregulation. Reduced expression of −1800/+65 APE1-Luc(0.5µg) + + + + + + + + both endogenous APE1 and APE1 promoter-dependent WTp53 (µg) −− 0.05 0.05 0.1 0.1 0.3 0.3 luciferase activity by exogenous p53 in unstressed p53 null TSA (100 ng/ml) − + − + − + − + cells further confirmed that p53 downregulates APE1 −177 −143 expression. However, ectopic expression of WTp53 down- APE1 5′ AGA GGA GGG AGG CGA GGC TAA GCG TCT CCG TCA CGT 3′ regulated the episomal APE1 promoter to a much greater probe extent than the endogenous gene which was somewhat unexpected. It is possible that the APE1 promoter in the Transcriptional chromatinized plasmid is more sensitive to the effect of HindIII (−128) start site AFlII CCAAT (+1) (+65) p53 than the folded conformation in cellular chromatin. −264 Furthermore, the endogenous APE1 gene could be regulated by binding of transcription factors to cis elements absent in the episomal promoter. The V143A AP-1 Nrul p53 mutant lacking DNA-binding activity did not down- GRE Sp1 USF (−173) regulate the basal APE1 promoter as much as did WTp53, Nuclear extract − ++ + + which strongly suggests that p53’s DNA-binding ability APE1 oligo. ++++ + is a prerequisite for its repressor activity. However, the WTp53 (µg) −− − 0.6 0.6 APE1 oligo. − −− + + L22G, T23S double mutant with normal DNA-binding activity, but lacking trans-acting activity (52), was also unable to repress the APE1 promoter, which suggests that both DNA binding and transactivation abilities of p53 are required for the repression. Western analysis showing that p53 mutants were more stable than the WTp53 protein eliminated the possibility that the former’s inability to repress the APE1 promoter was due to reduced stability. Figure 7. Effect of TSA on p53-mediated repression of APE1 promoter Using functional analysis of nested deletion mutants, (/) activity. (A) p53 cells were transiently transfected with 0.5mg we mapped the basal APE1 promoter to a 143 bp of APE1 promoter–luciferase plasmid (1800/+65) and increasing amounts of expression plasmid encoding WTp53 or equivalent amounts segment 5 to the transcription start site. We also observed of empty vector. Twenty-four hours after transfection cells were treated that the candidate p53-responsive cis element(s) were with or without TSA (100 ng/ml) and luciferase activity was measured located in the 184 to 143 bp segment. However, 48 h after transfection and normalized for the amount of protein. The sequence analysis revealed no apparent p53-binding sites mean  SD of five independent experiments performed in duplicate is within this segment. Moreover, the recombinant p53 shown. (B) Sequence of the DNA probes for EMSA assay with APE1 promoter. Schematic diagram of the APE1 promoter showing some of polypeptide failed to bind to an oligo of this sequence the putative transcription factor binding sites (Sp1; USF; glucocorticoid (184 to 143). At the same time we have provided direct response element (GRE); activator protein 1 (AP-1); and CCAAT box- evidence from ChIP assays that p53 was bound to the (/) like sequence). (C) EMSA with nuclear extracts of p53 cells 253 to 118 bp segment sequence in vivo. Together, these transfected with empty vector or WTp53 expression plasmid using P-labeled duplex oligo corresponding to bases 177 to 143 from data suggest that APE1 repression by p53 is not mediated APE1 promoter; P< 0.05. via direct cis element binding, but rather involves p53’s indirect recruitment to the promoter by other transcrip- tion factors. p53 has been shown to repress many other DISCUSSION promoters that lack p53-specific cis elements, indicating APE1 is often overexpressed in tumor cells compared to that the mechanisms of p53-mediated repression are its basal level in many untransformed cells lines. varied and complex (41,58,60,61). Such mechanisms Downregulation of APE1 was found to sensitize tumor could include (i) interference with transcriptional activa- cells to chemotherapy with induction of apoptosis tors, (ii) interference with the basal transcription machin- (26,27,29). Thus, elucidating the molecular basis for ery and (iii) compaction of the chromatin structure at the variable APE1 expression is important from both basic promoter sites by recruitment of HDACs and other and clinical perspectives. Although several studies have histone-modifying enzymes (41). shown that p53 participates in DNA repair processes, and The exact mechanism by which p53 represses the APE1 stimulates BER via its direct interaction with APE1 and expression is not clear. We have shown that candidate DNA polymerase b, whether p53 regulates APE1 level p53-responsive element(s) were located in the 184 to itself was not shown before (59). This report provides the 143 bp segment that includes one Sp1 site. An earlier first evidence that p53 downregulates APE1 expression. study using in vitro DNase I footprinting analysis with Fold inhibition 1564 Nucleic Acids Research, 2008, Vol. 36, No. 5 purified Sp1 polypeptide showed a distinct footprint CA98664 to T.I.), American Heart Association grant spanning 169 to 148 bp in the APE1 promoter (53). (AHA#0565008Y to K.B.). Amira Zaky was supported by Consistent with this, our ChIP assay showed constitutive a Government of Egypt scholarship for research at (+/+) Sp1 binding within this region in both p53 and University of Texas medical Branch. Funding to pay the (/) p53 cells, indicating that the Sp1 site is functional Open Access publication charges were waived by Oxford in vivo. Moreover, we observed that Sp1 remains bound to University Press. the APE1 promoter after p53 activation by CPT, Conflict of interest statement. None declared. suggesting that both p53 and Sp1 can simultaneously occupy the same promoter region. 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