The 3'-Untranslated Region of p21WAF1 mRNA Is a Composite cis-Acting Sequence Bound by RNA-binding Proteins from Breast Cancer Cells, Including HuR and Poly(C)-binding Protein*

Keith M. GilesDagger §, John M. DalyDagger , Dianne J. BeveridgeDagger , Andrew M. ThomsonDagger , Dominic C. VoonDagger , Henry M. Furneaux||, Jalal A. Jazayeri**, and Peter J. LeedmanDagger DaggerDagger

From the Dagger  Laboratory for Cancer Medicine and University Department of Medicine, Western Australian Institute for Medical Research and Centre for Medical Research, the University of Western Australia and ** Department of Endocrinology and Diabetes, Royal Perth Hospital, Perth, Western Australia 6001, Australia and || Vascular Biology Program, Department of Physiology, University of Connecticut Health Center, Farmington, Connecticut 06030

Received for publication, August 19, 2002, and in revised form, November 11, 2002

    ABSTRACT
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Despite promoting growth in many cell types, epidermal growth factor (EGF) induces growth inhibition in a variety of cancer cells that overexpress its receptor. The cyclin-dependent kinase inhibitor p21WAF1 is a central component of this pathway. We found in human MDA-468 breast cancer cells that EGF up-regulates p21WAF1 mRNA and protein, through a combination of increased mRNA stability and transcription. The decay rate of a hybrid luciferase reporter full-length p21WAF1 3'-untranslated region (UTR) mRNA was significantly faster than that of a control mRNA. Transfections with a variety of p21WAF1 3'-UTR constructs identified multiple cis-acting elements capable of reducing basal reporter activity. Short wavelength ultraviolet light induced reporter activity in constructs containing the 5' region of the p21WAF1 3'-UTR, whereas EGF induced reporter activity in constructs containing sequences 3' of the UVC-responsive region. These cis-elements bound multiple proteins from MDA-468 cells, including HuR and poly(C)-binding protein 1 (CP1). Immunoprecipitation studies confirmed that HuR and CP1 associate with p21WAF1 mRNA in MDA-468 cells. Over- and underexpression of HuR in MDA-468 cells did not affect EGF-induced p21WAF1 protein expression or growth inhibition. However, binding of HuR to its target 3'-UTR cis-element was regulated by UVC but not by EGF, suggesting that these stimuli modulate the stability of p21WAF1 mRNA via different mechanisms. We conclude that EGF-induced p21WAF1 protein expression is mediated largely by stabilization of p21WAF1 mRNA elicited via multiple 3'-UTR cis-elements. Although HuR binds at least one of these elements, it does not appear to be a major modulator of p21WAF1 expression or growth inhibition in this system. CP1 is a novel p21WAF1 mRNA-binding protein that may function cooperatively with other mRNA-binding proteins to regulate p21WAF1 mRNA stability.

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Inhibition of human tumor cell growth is mediated by a variety of cell cycle-related proteins and tumor suppressors. p53, a well characterized tumor suppressor, activates transcription of a number of target genes, including p21WAF1 (wild-type p53 activated fragment-1) (1, 2), which encodes a protein of Mr 21,000 (p21), also known as cyclin-dependent kinase-interacting protein 1. p21WAF1 inhibits cyclin-cyclin-dependent kinase activity, preventing phosphorylation of critical cyclin-dependent kinase substrates, blocking transition from G1 to S phase of the cell cycle (3), as well as inducing apoptosis (4). Recent evidence suggests that factors other than p53, such as EGF1 (16), can induce p21WAF1 expression in various cell types (p53-independent pathways). Because most human tumors lack p53 function (5), investigation of the mechanisms that regulate p21WAF1 expression through alternative growth factor-induced pathways has become an important focus in cancer research. In particular, a major goal is to devise approaches that would increase expression of p21WAF1 in tumors to reduce proliferation and tumor growth.

Although EGF is typically growth-proliferative in breast cancer cells (6), some cancer cells are growth-inhibited by EGF (e.g. MDA-468 breast (7, 8), A431 epidermoid (9, 10)). EGF-induced growth inhibition of these cells is associated with EGF receptor (EGFR) overexpression (11) and appears to be mediated by induction of p21WAF1 mRNA and protein (8). Multiple reports show conclusively that the regulation of p21WAF1 expression by growth factors and other ligands occurs predominantly at the level of mRNA stability (12-17). However, there is little understanding of the specific RNA-protein interactions involved in this process, particularly in breast cancer cells. Thus, the EGF-induced up-regulation of p21WAF1 mRNA provides an ideal system to investigate the mechanisms governing p21WAF1 mRNA decay.

The regulation of mRNA decay is a critical mechanism in the control of gene expression (reviewed in Hollams et al. (18)) that involves interactions between cis-acting sequences that confer instability to mRNA and the trans-acting protein factors that bind them. Many cis-acting sequences consist of AU-rich elements (AREs), most often located in the 3'-untranslated region (3'-UTR) of labile mRNAs. However, cis-acting elements are also found within the coding regions and 5'-UTRs of various mRNAs (e.g. c-fos, c-myc) (19). AREs often contain single or multiple repeats of pentamer (AUUUA) sequences, and inclusion of the AUUUA pentamer motif often targets the mRNA for rapid cytoplasmic degradation (20). One well characterized cis-acting element is the AU-rich sequence found within the 3'-UTR of granulocyte monocyte colony-stimulating factor mRNA, which is able to reduce the half-life of beta -globin mRNA from many hours to less than 30 min (20). Other studies have shown the UUAUUUA(A/)U(A/U) nonamer sequence to be more predictive of rapid mRNA decay than the AUUUA pentamer motif (21, 22). Several other RNA binding motifs have been identified, including the C-rich motif that is the target for the poly(C)-binding proteins (CPs) (23). To date, the cis-activity of the 3'-UTR of p21WAF1 has not been characterized extensively.

Multiple proteins have been identified that can bind to AU- and U-rich regions (reviewed in Hollams et al. (18)). These include AUBF (24), AUF1 (hnRNP D) (25), Hel-N1 (26), hnRNP C (27), hnRNP A1 (27), AUH (28), HuR (29), HuD (30), tristetraprolin (31), and poly(A)-binding protein (32). Of the AU- and U-rich-binding proteins, only a few have been shown to definitively regulate mRNA stability: AUF1, HuR, and other Hu/ELAV proteins and tristetraprolin and its family members.

HuR, a ubiquitously expressed member of the Hu/ELAV family, is involved in the shuttling of transcripts from the nucleus into the cytoplasm (33-35), as well as in the regulation of mRNA stability (17, 35-38). In RKO colorectal carcinoma cells, HuR mediates UVC-induced stabilization of p21WAF1 mRNA (17), and of interest, HuD, a neuron-specific member of the Hu/ELAV family (30), has been shown to bind to a 42-nt sequence within the 3'-UTR of p21WAF1 mRNA (39). It seemed possible, therefore, that HuR would play an important role in the regulation of p21WAF1 mRNA stability in breast cancer cells.

Here, we show that the 3'-UTR of p21WAF1 mRNA contains multiple cis-acting regions that reduce basal reporter activity and confer EGF- and UVC-induced changes to reporter constructs in a region-specific manner. These 3'-UTR elements are the target for a number of RNA-binding proteins, including HuR and CP1, from MDA-468 breast cancer cells. Despite its role in the mediation of p21WAF1 mRNA stabilization and p21WAF1 expression by UVC in other cell systems, HuR does not appear to have a major role in EGF-induced p21WAF1 expression in MDA-468 breast cancer cells (EGFR overexpressed, mutant p53).

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Cell Culture-- The MDA-468 (HTB 132) human breast cancer cell line was obtained from ATCC (Manassas, VA). Cells were routinely cultured in Dulbecco's modified Eagle's medium/F-12 medium supplemented with 10% fetal calf serum (Invitrogen). BING cells (40) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. All cell lines were cultured in the presence of penicillin (50 units/ml) and streptomycin (50 µg/ml). Cells were utilized within 12 passages of the original stock received from ATCC for all experiments.

Cell Cycle Analysis-- For cell cycle analysis, EGF-treated (25 ng/ml, 4 nM) and control MDA-468 cells were harvested by trypsinization and then permeabilized and stained for DNA in phosphate-buffered saline (PBS) containing 0.1% Nonidet P-40, 5 mM EDTA, 5 mM EGTA, 5 µg/ml propidium iodide, and 100 µg/ml RNase A. Flow cytometry was performed on a Coulter EPICS XL-MCL (Coulter Corp., Hialeah, FL), and cell cycle analysis was performed with MultiPlus AV MultiParameter data analysis software (Phoenix Flow Systems, San Diego, CA).

Plasmid Clones, cDNA Probes, Riboprobes, Fusion Proteins, and Expression Clones-- The p21WAF1 plasmid cDNA (pCEP-WAF1) (from Dr. B. Vogelstein) contained the 5'-UTR, coding region, and 3'-UTR of p21WAF1 (see Fig. 1A; nucleotide sequence is in the GenBankTM data base under accession number U03106) (1) and was digested with EcoRI and NotI to liberate a 1-kb cDNA fragment, which for Northern analysis, was random prime-labeled using [32P]dCTP (~3000 Ci/mmol; Amersham Biosciences). A 1.1-kb 18 S rRNA cDNA probe was used as a loading control. Plasmids WAF1-1/7, WAF1-2/7, WAF1-6/7, WAF1-879, WAF1-1512, and WAF1-1/6 (Fig. 1A) were constructed by cloning PCR-amplified sequences from the 3'-UTR of the p21WAF1 cDNA into either the XbaI site of pGL3-control luciferase reporter vector (Promega) for transfection experiments or into the BamHI/HindIII-digested pBluescript II KS+ vector (Stratagene) for the generation of labeled riboprobes. The plasmid containing the HuD binding site (WAF1-HuD) were constructed by subcloning annealed 42-mer sense (nt 657-698, 5'-UCU UAA UUA UUA UUU GTG UUU UAA UUU AAA CAC CUC CUC AUG-3') (39) and antisense oligonucleotides corresponding to this region of p21WAF1 3'-UTR (see Fig. 1, A and B) into the BamHI/HindIII sites of the pBluescript vector. The c-fos AU-rich element that generates an unstable mRNA was also cloned into the XbaI site of pGL3-control and used in transfection assays (22). pRL-SV40 (Promega) was utilized as a control for transfection efficiency in reporter assays. Some plasmids contained three AU-rich sequences (shown in Fig. 1, A and B) and are denoted A (nt 742-758, 5'-AAU UAU UUA AAC AAA AA-3'), B (nt 797-809, 5'-AUU UUU AUU UUA U-3'), and C (nt 811-824, 5'-AAA UAC UAU UUA AA-3'). For some RNA gel shifts (RNA electrophoretic mobility shift assays) the plasmid c-fos-HuD (5'-AUA UUU AUA UUU UUA UUU UAU UUU UUU-3') (29) was also used (Fig. 1B). All pBluescript plasmid clones were linearized with HindIII for transcription with T7 RNA polymerase (Invitrogen) in reactions containing [32P]UTP (3000 Ci/mmol; Amersham Biosciences), as described (41), to produce riboprobes with a specific activity of ~2 × 109 cpm/µg RNA that included 66 nt of pBluescript, in addition to the corresponding portion of the p21WAF1 3'-UTR. Unlabeled RNA transcripts were synthesized as above except with 2.5 mM rNTPs, quantified by spectrophotometry and verified by PAGE. pGEX-2T-HuR (from Dr. H. Furneaux) generated a fusion protein (GST-HuR) that contained amino acids 2-326 of human HuR (29). pGEX-6P-alpha CP1 (from Dr. M. Kiledjian) generated a fusion protein (GST-CP1) that contained amino acids 13-347 of human CP1 (60). For HuR over-/underexpression studies, the retroviral vector pBabe puro (42) was used. The sequence of all plasmid constructs was confirmed by dideoxy sequencing.


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Fig. 1.   Schematic of p21WAF1 3'-UTR and clones used in vitro. A, schematic representation of the p21WAF1 mRNA 3'-UTR sequence and clones used for transfection (pGL3) and REMSA (pBluescript). The 42-nt HuD binding sequence (39) and three AU-rich regions (A, B, and C) are delineated, together with a number of C-rich motifs (shown beneath the 3'-UTR sequence, denoted C). The numbers refer to nucleotide positions within the p21WAF1 mRNA sequence (GenBankTM accession number U03106). B, sequences of c-fos HuD (nt 3399-3425 of GenBankTM accession number V01512), WAF1-HuD (nt 657-698 of GenBankTM accession number U03106), and WAF1-1/6 (nt 571-829 of GenBankTM accession number U03106). Within WAF1-1/6, WAF1-HuD is underlined and italicized, a CCUCC consensus motif for CP1 is shown in bold italics, and the three AU-rich sequences, A, B, and C, are shaded. C, schematic representation of the GST fusion proteins used for in vitro assays. HuR contains three RNA recognition motif domains (RRMI, RRMII, RRMIII). CP1 contains three K-homology domains (KHI, KHII, KHIII). RNA-binding domains are shown to scale, and the amino acid boundaries of each are defined.

RNA Isolation and Northern Analysis-- MDA-468 cells were solubilized in 4 M guanidinium isothiocyanate, and total RNA was isolated using the method of Chomczynski and Sacchi (43). RNA (10-15 µg per sample) was size-fractionated on a 1% agarose-formaldehyde gel and transferred to Hybond-N+ membrane (Amersham Biosciences). RNA was UV cross-linked to the membrane, which was prehybridized for 4 h at 42 °C in a buffer containing 50% formamide, 0.75 M NaCl, 0.075 M sodium citrate, pH 7.0, 5× Denhardt's solution, 1% SDS, and 200 µg/ml salmon sperm DNA and then hybridized in the same buffer overnight at 42 °C with 32P-labeled p21WAF1 cDNA probe at 106 cpm/ml. The membrane was washed sequentially in 2× SSC/0.1% SDS for 20 min at 22 °C, 0.2× SSC/0.1% SDS for 20 min at 22 °C, and finally in 0.2× SSC/0.1% SDS at 65 °C for 5 min. Membranes were imaged with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and quantified using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). In all experiments an 18 S rRNA cDNA probe was used for normalization.

mRNA Turnover Studies-- MDA-468 cells (70-80% confluent) were treated with EGF (25 ng/ml) (Promega) or cycloheximide (10 µg/ml) for 2 h followed by the addition of the transcription inhibitor actinomycin D (ActD) at 7.5 µg/ml (Sigma). Total RNA was isolated from the cells at 0-, 2-, 4-, and 8-h time intervals after addition of ActD and subjected to Northern analysis as described earlier. p21WAF1 mRNA half-life was determined using linear regression analysis.

Nuclear Run-on Transcription Assay-- MDA-468 cells (70-80% confluent) were treated with EGF (25 ng/ml) for 2 h. Nuclei were isolated as described previously (44), rapidly frozen, and stored at -85 °C. The transcription assay was performed as described previously (45). Briefly, the nuclei were thawed on ice, resuspended in 100 µl of reaction buffer (10 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 300 mM KCl, 5 mM dithiothreitol (DTT), 0.5 mM each of ATP, CTP, and GTP, and 100 µCi of [32P]UTP (3000 Ci/mmol; Amersham Biosciences)), and incubated at 30 °C for 30 min. Labeled RNA was isolated and hybridized to nitrocellulose filters onto which 5 µg of p21WAF1 and 18 S rRNA cDNAs had been blotted. Filters were washed and then analyzed by PhosphorImager and ImageQuant software.

Immunoblot Assay for p21WAF1, HuR, and Actin Protein-- Control and EGF (25 ng/ml)-treated MDA-468 cells were harvested and lysed in ice-cold radioimmune precipitation assay lysis buffer (1% Nonidet P-40, 0.1% SDS, 0.5% deoxycholate, 150 mM NaCl, 50 mM NaF, 1 mM DTT, 50 mM Tris, pH 8.0), containing freshly added protease inhibitors (1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml leupeptin, 2 µg/ml aprotinin (Roche Molecular Biochemicals)). After 10 min on ice, the lysate was centrifuged at 750 × g for 10 min at 4 °C, after which the supernatant was recovered and stored at -85 °C. Total protein concentrations of lysates were determined using the Bio-Rad protein assay, and 10 µg of proteins were separated on 10% BisTris acrylamide gels (Invitrogen) in 1× MES SDS running buffer, pH 7.3 (Invitrogen), and transferred to polyvinylidene difluoride membranes (Osmonics) in 1× NuPAGE transfer buffer, pH 7.2, according to the manufacturer's instructions. Membranes were blocked with 10% skim milk in TBS-T (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) at 22 °C for 1 h, prior to addition of either anti-p21WAF1 monoclonal antibody (1:1000) (15091A; BD Biosciences), anti-HuR monoclonal antibody 19F12 (1:2000) (from Henry Furneaux), or anti-actin monoclonal antibody (1:2000) (I-19, sc-1616; Santa Cruz Biotechnology, Santa Cruz, CA), diluted in 10% skim milk/TBS-T, for 1 h at 22 °C. Membranes were then washed in 10% skim milk/TBS-T, incubated for 1 h in appropriate peroxidase-conjugated secondary antibody (1:10000; Amersham Biosciences), and washed again with TBS-T, prior to detection with ECL Plus detection reagents (Amersham Biosciences) on ECL-Hyperfilm (Amersham Biosciences). Protein bands were quantified using an Eastman Kodak Co. digital DCS-420C camera and ImageQuant software.

Real-time PCR Assay for Luciferase mRNA Decay-- MDA-468 cells (50% confluent) were transiently transfected with 8 µg of pGL3-control, pGL3-WAF1-1/7, or pGL3-c-fos ARE using FuGENE (Roche Molecular Biochemicals), according to the manufacturer's instructions. The cells were passaged, and 38 h after transfection they were treated with ActD (7.5 µg/ml) (Sigma) for 0-4 h. Total MDA-468 RNA was harvested using TRIzol (Invitrogen). To generate cDNA, 2 µl of RNA (denatured at 70 °C for 10 min) was reverse-transcribed in a 20-µl reaction containing 5 mM MgCl2, 1× avian myeloblastosis virus reverse transcriptase buffer, 1 mM dNTPs, 20 units of RNasin, 10 units of avian myeloblastosis virus reverse transcriptase, and 250 ng of oligo(dT)15 primer at 42 °C for 30 min. PCR was then performed for both luciferase and beta -actin cDNA (luciferase sense, 5'-TAC TGG GAC GAA GAC GAA CAC-3'; luciferase antisense, 5'-GTT CAC CGG CGT CAT CGT CG-3'; beta -actin sense, 5'-GCC AAC ACA GTG CTG TCT GG-3'; beta -actin antisense, 5'-TAC TCC TGC TTG CTG ATC CA-3') using a Bio-Rad iCycler iQ real-time PCR detection system (Bio-Rad). Data were normalized using results obtained for beta -actin and the ratio of luciferase mRNA remaining for pGL3-WAF1-1/7 and pGL3-c-fos-ARE expressed relative to pGL3-control as a function of time after ActD treatment.

Transfection and Luciferase Assays-- MDA-468 cells (50% confluent) were transiently transfected with 8 µg of pGL3 ± various p21WAF1 3'-UTR regions (see Fig. 1A) and 100 ng of pRL-SV40 as a control, using FuGENE 6 as above. Some cells were cultured following treatment with EGF (25 ng/ml) or UVC (254 nM, 20 J/m2), for 8 or 6 h respectively, prior to lysate extraction. Cells were washed in PBS, harvested by trypsinization, and lysed, and supernatant luciferase activity was measured using the dual luciferase reporter assay kit (Promega) and a Wallac Victor 1420 multilabel counter (Wallac Oy; Turku, Finland), according to the manufacturer's instructions. Firefly luciferase (pGL3) activity was normalized against Renilla luciferase (pRL-SV40) activity to yield the relative luciferase activity.

Preparation of Cytoplasmic Extracts for RNA Gel Shift Assays-- MDA-468 cells were grown to 70-80% confluence in 10-cm culture dishes. Cytoplasmic extracts were prepared as described previously (41). Briefly, cells were scraped from the culture dishes in chilled PBS, centrifuged at 450 × g for 4 min at 4 °C, washed again with PBS, and then incubated for 20 min with cold cytoplasmic extract buffer (CEB; 10 mM HEPES, 3 µM MgCl2, 40 mM KCl, 5% glycerol, 0.2% Nonidet P-40, 1 mM DTT), containing freshly added protease inhibitors (0.5 mM PMSF, 10 µg/ml leupeptin, 2 µg/ml aprotinin). Lysates were cleared by centrifugation at 4 °C for 10 min at 12,100 × g, and the supernatant was snap-frozen in liquid nitrogen and stored at -80 °C. Protein concentrations were determined using the Bio-Rad protein assay kit.

Preparation of Whole Cell Extracts for RNA Gel Shift Assays-- MDA-468 cells were grown to 70-80% confluence in 10-cm culture dishes. Medium was removed, the cell monolayer was washed twice in ice-cold PBS, and the cells were lysed in 0.5 ml of chilled lysis buffer (containing 50 mM Tris, pH 7.5, 5 mM EDTA, pH 8.5, 150 mM NaCl, 1% Triton X-100, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM PMSF, 2 mM NaVO4, 50 mM NaF, and 10 mM Na2MoO4·2H2O) on ice for 10 min. Cells were scraped and transferred to Eppendorf tubes, and lysates were then cleared by centrifugation at 4 °C for 10 min at 12,100 × g and stored at -80 °C. Protein concentrations were determined by Bio-Rad protein assay kit.

RNA Electrophoretic Mobility Shift Assay (REMSA)-- Binding reactions were performed as described previously (41) with 5 µg of cytoplasmic extract or 200 ng of recombinant protein and 105 cpm of 32P-labeled RNA (~2-5 pg). Briefly, binding reactions were incubated at 22 °C for 30 min, after which 0.3 units of RNase T1 (Roche Molecular Biochemicals) was added for 10 min, followed by the addition of heparin (final concentration 5 mg/ml) (Sigma) for 10 min. Complexes were separated by 4% PAGE, visualized by PhosphorImager, and analyzed by ImageQuant software (Molecular Dynamics, Sunnyvale, CA). In competition assays, extracts were incubated with an unlabeled competitor RNA (~100-fold excess) for 30 min prior to addition of the 32P riboprobe. For supershift assays with the HuR antibody, the method used was as described previously (46).

UV Cross-linking (UVXL) of RNA-Protein Complexes-- RNA-protein binding reactions were carried out as described above, using 20 µg of whole cell extract or 100-500 ng of recombinant protein, 1.5 × 105 cpm (10-15 pg) of 32P riboprobe (41), and 2-5 µg of tRNA. Following the addition of heparin, samples were placed on ice in a microtiter tray and UV-irradiated 1 cm below the Stratalinker UV light source (240 nm UV-bulb; Stratagene) for 10 min. After UVXL, samples were incubated with RNase A (final concentration 100 µg/ml) (Roche Molecular Biochemicals) at 37 °C for 15 min. The samples were boiled for 3 min in SDS sample buffer (50% glycerol, 0.25 M Tris, pH 6.8, 10% SDS, 4% beta -mercaptoethanol) and subjected to SDS-PAGE (gels ranging from 8.5% to 12%), and RNA-protein complexes were detected by PhosphorImager. In some competition experiments, recombinant proteins were incubated with ribohomopolymers (poly(C), poly(A)) for 20 min, prior to addition of riboprobe. For UVXL immunoprecipitation assays, UVXL was performed as described above, and the reactions were then incubated for 1 h with HuR or GST antibody at 4 °C, followed by incubation with protein A and G beads (Sigma) for 45 min at 4 °C. After washing, RNA-protein complexes were resolved by SDS-PAGE (gels ranging from 8.5 to 12%) and detected by PhosphorImager. In all UVXL experiments, 14C Rainbow molecular mass markers (Amersham Biosciences) were used.

Preparation of GST Fusion Proteins-- GST fusion proteins were prepared essentially as described (47). Briefly, 1-liter cultures of DH5alpha Escherichia coli expressing GST, GST-HuR, or GST-CP1 fusion constructs (described above) were induced at A600 of 0.6 with isopropyl-beta -D-thiogalactopyranoside (0.5 mM) at 30 °C for 2 h. GST fusion protein was purified from bacterial pellets, lysed in 10 mM Tris, pH 7.8, 0.5 mM EDTA, 100 mM glucose, 0.4 mg/ml lysozyme, 0.13% Triton X-100, 0.5 mM PMSF, 1 µg/ml aprotinin, and 1 µg/ml leupeptin, using glutathione beads (Sigma). GST protein was eluted in buffer containing 20 mM HEPES, pH 7.6, 100 mM KCl, 0.2 mM EDTA, 20% glycerol, 1 mM DTT, 0.5 mM PMSF, 1 µg/ml aprotinin, 2 µg/ml leupeptin, and 25 mM glutathione. To cleave HuR from the GST-HuR fusion protein, 0.1 units/µl of thrombin (Amersham Biosciences) was incubated with the GST-HuR on glutathione beads overnight at 4 °C, the sample was centrifuged at 12,100 × g at 4 °C for 2 min, and the supernatant was collected. CP1 was cleaved from GST-CP1 with PreScission protease (Amersham Biosciences), according to the manufacturer's instructions. Purified proteins were quantified by Bio-Rad protein assay, and purity was ascertained by SDS-PAGE.

IP-RT-PCR Assay-- MDA-468 cells were grown to 50% confluence in 10-cm dishes, and cytoplasmic extracts were harvested as described above. Cytoplasmic extract (200 µg) was added to 10 µg of HuR, CP1, or EGFR antibody. After incubation on ice for 45 min, 5 µg each of protein A (Amersham Biosciences) and protein G (Sigma) beads was added to all tubes, which were mixed for a further 45 min at 4 °C. The tubes were centrifuged at 2,000 × g for 2 min, and the supernatants were removed for RNA extraction. The pelleted beads were washed with cold CEB (10 × 1 ml), and RNA was extracted using TRIzol reagent. Reverse transcription was performed using random hexamers (Promega) and standard procedures. PCR was performed for 33 cycles, comprising five cycles of denaturation at 94 °C/30 s, annealing at 66 °C/30 s, and extension at 72 °C/1 min, followed by 28 cycles of denaturation at 94 °C/30 s, annealing at 55 °C/30 s, and extension at 72 °C/1 min, with the primers 481F (5'-GAC TCT CAG GGT CGA AAA CG-3') and 585R (5'-CTT CCT GTG GGC GGA TTA G-3') from within the coding sequence of p21WAF1. These primers produce an amplicon that spans an intron, allowing the discrimination between cDNA- and genomic DNA-related products in these PCR experiments. PCR products were resolved on an ethidium bromide-stained 3% agarose gel.

Retroviral Expression of HuR-- Full-length HuR cDNA was cloned into the pBabe puro vector (42) in the sense and antisense orientation and then transiently transfected into the retroviral packaging cell line BING (40) using FuGENE according to the manufacturer's protocol. Retroviral-containing conditioned medium was collected from the BING cells at ~48 h after transfection. Following filtration (0.45 µm) and the addition of 4 µg/ml polybrene (hexadimethrine bromide; Sigma), the retroviral-containing medium was incubated overnight with the target cells (MDA-468). Cells were selected in 1 µg/ml puromycin (Sigma) starting 48 h after infection. Pools of puromycin-resistant cells were analyzed by Western blotting to confirm transgene expression. All subsequent experiments were performed using pools of infected cells.

Colony Formation Assays-- Colony formation assays were performed as described previously (58). Briefly, MDA-468 sublines were plated in triplicate at a density of 5000 cells per 10-cm plate and then incubated overnight. A single dose of EGF (25 ng/ml) or PBS control was added at 24 h, and the cells were allowed to grow for a further 10 days. After fixation in methanol:acetic acid (3:1), colonies were stained with Giemsa (Fluka) and counted using a Quantimet 520 image analyzer (Leica).

Statistical Analysis-- Transfection and luciferase mRNA decay data are shown as mean ± S.D. Statistical analysis was performed using Student's t test, with a p value of <0.05 regarded as significant.

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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

EGF Up-regulates p21WAF1 Expression in MDA-468 Cells-- The MDA-468 breast cancer cell line, which contains mutant p53 and overexpresses the EGFR (7), provides an excellent model system to investigate the mechanisms underlying p21WAF1 gene expression and its regulation by EGF. To establish the validity of this cell line as a model of EGF-induced cell cycle arrest and growth inhibition, the cells were treated with EGF (25 ng/ml) for 8 h, and the proportion of cells in S-phase of the cell cycle was determined using flow cytometry (see "Materials and Methods"). EGF treatment reduced the proportion of cells in S phase from 30 to 12.5% (data not shown). Furthermore, in a colony formation assay (see "Materials and Methods"), EGF treatment led to >98% reduction in the number of detectable colonies (data not shown).

We next examined the effect of EGF on endogenous p21WAF1 mRNA and protein levels in MDA-468 cells. EGF rapidly up-regulated p21WAF1 protein (Fig. 2A) and p21WAF1 mRNA (Fig. 2B) within 2 h. To determine whether this up-regulation of p21WAF1 mRNA occurred at the post-transcriptional level, we treated MDA-468 cells with EGF for 2 h and performed ActD chase studies. In the absence of EGF, the basal half-life of p21WAF1 mRNA was relatively short (2.7 h) (Fig. 2C). However, in the presence of EGF the half-life was increased to 11.5 h (Fig. 2C). Treatment of the cells with cycloheximide, a translational inhibitor, also stabilized p21WAF1 mRNA (Fig. 2C), suggesting that (i) ongoing translation is required for maintenance of the short basal half-life, and/or (ii) existing cellular proteins mediate the stabilization of p21WAF1 mRNA.


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Fig. 2.   EGF increases p21WAF1 mRNA and protein levels and p21WAF1 mRNA stability and transcription in MDA-468 cells. A, Western blot analysis showing p21WAF1 protein levels in MDA-468 cells following treatment with EGF (25 ng/ml). Cell lysates (10 µg) were subjected to SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with anti-p21WAF1 and actin antibodies. ECL-generated images were quantified using ImageQuant and p21WAF1 protein levels graphed over time. B, Northern blot analysis of total RNA extracted from MDA-468 cells after treatment with EGF (25 ng/ml) for the times indicated. Following hybridization with a 32P-labeled p21WAF1 cDNA probe, each blot was normalized using a 32P-labeled 18 S ribosomal RNA cDNA probe. Quantification was performed using a PhosphorImager and ImageQuant software, and p21WAF1 mRNA levels were graphed over time. C, ActD chase studies in MDA-468 cells. Cells were grown to 50% confluence and treated with 25 ng/ml EGF or 10 µg/ml cycloheximide (CHX) for 2 h and then 7.5 µg/ml ActD. Total RNA was extracted from the cells at 0, 2, 4, or 8 h after ActD treatment and analyzed by Northern blot as in B. p21WAF1 mRNA was normalized against 18 S rRNA (image not shown). Half-life of p21WAF1 mRNA was 11.5 h (EGF-treated cells) and 2.7 h (control cells). **, p < 0.01. D, transcription run-on analysis of MDA-468 cells after treatment with EGF (25 ng/ml) for 2 h. p21WAF1 transcription rates were measured in isolated nuclei by run-on transcription assays, and the results were analyzed by PhosphorImager and ImageQuant and shown in this figure relative to 18 S rRNA transcription levels. CON, control; EGF, 2-h EGF treatment.

Nuclear run-on assays were employed to evaluate the effect of EGF on p21WAF1 transcriptional activity. After treatment of MDA-468 cells with EGF, p21WAF1 transcription increased by ~2-fold (Fig. 2D). Taken together, these data suggest that the EGF-induced growth arrest in MDA-468 cells is associated with a rapid increase in p21WAF1 mRNA and protein, which results from a combination of increased mRNA stability and increased transcription.

Identification of cis-Acting Elements in the 3'-UTR of p21WAF1 mRNA-- Based on the observation that the stabilization of p21WAF1 mRNA is a major contributor to the overall up-regulation of p21WAF1 protein expression in EGF-treated MDA-468 cells, we next sought to elucidate some of the mechanisms underlying this effect. In breast cancer cells, little is known of the cis-acting elements or trans-acting factors involved in the regulation of p21WAF1 mRNA stability. Transfection studies with ActD chase and real-time PCR demonstrated that WAF1-1/7 (entire p21WAF1 3'-UTR; see Fig. 1A) destabilized luciferase mRNA significantly, in a manner equivalent to that of the highly unstable c-fos ARE (Fig. 3). These data provided strong evidence that the p21WAF1 3'-UTR contains one or more cis-elements that confer basal mRNA instability and potentially contribute to the regulation of p21WAF1 mRNA stability.


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Fig. 3.   The 3'-UTR of p21WAF1 mRNA is a cis-acting sequence. MDA-468 cells were transfected with 8 µg of pGL3-control, pGL3-WAF1-1/7 (see Fig. 1), or pGL3-c-fos ARE, and 38 h after transfection they were treated with ActD (7.5 µg/ml) for 0, 2, and 4 h. Total MDA-468 RNA was harvested, and cDNA was generated by RT and used in real-time PCR with luciferase- and beta -actin-specific primers. Data were normalized using results obtained for beta -actin, and the ratio of luciferase RNA remaining for pGL3-WAF1-1/7 and pGL3-c-fos-ARE was expressed relative to pGL3-control. The data are representative of three separate experiments performed in triplicate. **, p < 0.001.

The 3'-UTR of p21WAF1 contains an AU-rich region at the 5' end that spans ~250 nt, termed WAF1-1/6 (see Fig. 1, A and B), which contains at least one known HuR binding site (17). Within WAF1-1/6 is a 42-nt sequence, termed WAF1-HuD, which contains an imperfect consensus nonamer and is the target for HuD binding (39). The WAF1-1/6 region also contains several smaller stretches of AU-rich sequence, denoted A, B, and C (see Fig. 1, A and B, and see "Materials and Methods") and was therefore a candidate cis-acting sequence.

To further dissect the cis-activity of the p21WAF1 3'-UTR, we generated several reporter constructs containing portions of the 3'-UTR of p21WAF1 (Fig. 1A). In transfection assays using MDA-468 cells, the full-length 3'-UTR, WAF1-1/7, reduced basal reporter activity by ~85% (Fig. 4, A and B), supporting our ActD-luciferase mRNA data (Fig. 3). Subsequent analysis of the three major components of the 3'-UTR (WAF1-1/6, WAF1-879, WAF1-1512) showed that each reduced reporter activity but that the major effect was 3' of the previously identified AU-rich region contained within WAF1-1/6 (Fig. 4B). In support of this observation, clones WAF1-2/7 and WAF1-6/7, which harbored deletions of the AU-rich regions, reduced reporter activity similarly to WAF1-1/7. Taken together, these results suggest that the WAF1-1/6 region is not the sole determinant of basal p21WAF1 mRNA stability in MDA-468 cells and that the four AU-rich regions (HuD; see Fig. 1, A-C) are not major contributors to basal turnover of p21WAF1 mRNA in MDA-468 cells. Furthermore, although WAF1-879 and WAF1-1512 each decrease reporter activity, neither is as effective as the combined sequence (WAF1-6/7) (Fig. 4B). This suggests the presence of multiple cis-elements within the 3'-UTR of p21WAF1 mRNA.


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Fig. 4.   The 3'-UTR of p21WAF1 mRNA contains elements that modulate basal luciferase reporter activity and confer UVC and EGF inducibility to reporter constructs. A, MDA-468 cells were transfected with 8 µg of pGL3 ± p21WAF1 3'-UTR elements and 100 ng of pRL-SV40 as a control. Cells were cultured in the presence or absence of EGF (25 ng/ml) or UVC (20 J/m2) treatment. Firefly and Renilla luciferase activity was measured as described under "Materials and Methods." B, luciferase reporter activity for MDA-468 cells transfected with pGL3 ± p21WAF1 3'-UTR elements expressed relative to pGL3-control. *, p < 0.002. C, luciferase reporter activity for EGF-treated MDA-468 cells transfected with pGL3 ± p21WAF1 3'-UTR elements. Values were expressed relative to untreated cells transfected with the same cis-element (untreated self). *, p < 0.002; **, p < 0.03. D, luciferase reporter activity for UVC-treated MDA-468 cells transfected with pGL3 ± p21WAF1 3'-UTR elements. Values were expressed relative to untreated cells transfected with the same cis-element (untreated self). *, p < 0.001; **, p < 0.02. The graphs are representative of at least three separate experiments, each performed in triplicate. All values were normalized using Renilla luciferase activity.

We next examined the effect of EGF on the reporter activity of each of these constructs in MDA-468 cells. EGF increased reporter activity by ~60% in the case of WAF1-1/7, with most of this effect being contained within the WAF1-6/7 region (Fig. 4C). Both the WAF1-879 and WAF1-1512 constructs conferred EGF-induced up-regulation of luciferase reporter activity, whereas the WAF1-1/6 construct appeared to be relatively EGF-unresponsive (Fig. 4C). Taken together, these results suggest that the predominant cis-element(s) within the 3'-UTR of p21WAF1 that are responsible for both basal mRNA instability and EGF-inducibility in MDA-468 cells reside downstream of the WAF1-1/6 sequence.

To compare these results with another regulator of p21WAF1 mRNA stability, we tested the effect of UVC treatment on reporter activity using the same constructs in transfection experiments with MDA-468 cells. The full-length 3'-UTR (WAF1-1/7) increased reporter activity by ~70% after UVC treatment (Fig. 4D). Further analysis revealed that the UVC-mediated up-regulation of reporter activity occurred predominantly through sequences contained within the WAF1-1/6 construct, with a lesser contribution from sequences downstream of WAF1-1/6 (Fig. 4D). This result suggests that the WAF1-1/6 region is the major 3'-UTR determinant of UVC-induced stabilization of p21WAF1 mRNA in MDA-468 cells. These data illustrate that even within the one cell type, different stimuli may lead to the preferential regulation of different and specific cis-elements within the p21WAF1 3'-UTR, presumably via different sets of RNA-protein interactions.

MDA-468 Cells Contain Proteins That Bind Specifically to p21WAF1 mRNA-- To investigate whether the cis-acting p21WAF1 mRNA 3'-UTR elements were a target for cytoplasmic RNA-binding proteins from MDA-468 breast cancer cells, we tested each region (WAF1-1/6, WAF1-HuD, WAF1-879, WAF1-1512) with REMSA. Cytoplasmic proteins from MDA-468 cells bound specifically to these probes (Fig. 5A, lanes 2, 6, 8, and 10), whereas no RNA-protein complexes were observed with 32P-labeled vector control (pBluescript; see Fig. 5A, lane 12). Furthermore, addition of ~100-fold molar excess unlabeled pBluescript competitor RNA did not diminish the formation of the RNA-protein complexes significantly (WAF1-HuD; see Fig. 5B, lane 4) (data not shown for the other riboprobes). However, addition of excess unlabeled self RNA (WAF1-HuD; see Fig. 5B, lane 3) (data not shown for the other riboprobes) virtually abolished RNA-protein complexes in all cases, demonstrating the specificity of the interaction.


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Fig. 5.   Proteins from MDA-468 cells bind specifically to cis-acting elements from the 3'-UTR of p21WAF1 mRNA. MDA-468 extracts were incubated with a panel of 32P-labeled p21WAF1 or vector control (pBluescript) riboprobes, and REMSA (4% PAGE) or UVXL (10 min of UVXL exposure, SDS-PAGE 8.5 to 12%) was performed as described under "Materials and Methods." A, REMSA utilizing various p21WAF1 (lanes 1, 2, and 5-10), vector control (pBluescript) (lanes 11 and 12), or c-fos HuD (lanes 3 and 4) riboprobes in the presence or absence of MDA-468 cytoplasmic extract. B, REMSA with WAF1-HuD riboprobe and MDA-468 extracts in the absence of competitor RNA (lane 2) and in the presence of unlabeled self RNA (lane 3) or unlabeled vector control RNA (lane 4). C, UVXL with MDA-468 cell extracts and WAF1-1/6 (lane 1) or WAF1-HuD (lane 3) riboprobes, and compared to 14C molecular mass markers (lane 2). D, UVXL with MDA-468 cell extracts and WAF1-879 (lane 2) and WAF1-1512 (lane 3) riboprobes; 14C molecular mass markers (lane 1).

We next utilized UVXL assays to characterize the individual p21WAF1 RNA-binding proteins. We found that multiple proteins targeted the WAF1-1/6, WAF1-HuD, WAF1-879, and WAF1-1512 probes (Fig. 5C, lanes 1 and 3; Fig. 5D, lanes 2 and 3, respectively). A similar set of RNA-protein complexes (RPCs) was identified with the WAF1-1/6 and WAF1-HuD probes, but the relative intensity of most of the RPCs was significantly different between the two probes (Fig. 5C, lanes 1 and 3). This suggested that the majority of these proteins bind to the WAF1-HuD region, which is contained within WAF1-1/6. Interestingly, a smaller range of RPCs was detected with the WAF1-879 and WAF1-1512 probes (Fig. 5D, lanes 2 and 3), and again the relative intensities of the RPCs differed between the two probes.

HuR and CP1 Bind to p21WAF1 mRNA-- Previous studies in other cell types have demonstrated that the WAF1-HuD region within p21WAF1 mRNA is a target for members of the ELAV RNA-binding protein family (e.g. HuD) (39). Wang et al. (17) showed that increased binding of HuR to the WAF1-1/6 element mediated the UVC-induced stabilization of p21WAF1 mRNA (17). This suggested that the ~36-kDa band observed in UVXL studies with the WAF1-HuD and WAF1-1/6 probes and using MDA-468 breast cancer extracts (Fig. 5C, lanes 1 and 3) contained HuR. In addition, a preponderance of potential CP1 binding sites within the p21WAF1 3'-UTR (see Fig. 1A), together with the observed RPCs at ~42 kDa using the p21WAF1 3'-UTR riboprobes (Fig. 5C, lanes 1 and 3; Fig. 5D, lanes 2 and 3), suggested that CP1 protein might target p21WAF1 mRNA.

To investigate the association among HuR, CP1, and p21WAF1 mRNA in MDA-468 cells, we utilized an immunoprecipitation-RT-PCR assay with primers that target the p21WAF1 coding region (see "Materials and Methods"). Using HuR and CP1 antibodies, we were able to co-immunoprecipitate p21WAF1 mRNA from MDA-468 cells (Fig. 6A, lanes 2 and 3). However, no p21WAF1-specific PCR product was identified when using an unrelated antibody (EGFR) (Fig. 6A, lane 4) or with no antibody (beads alone) (Fig. 6A, lane 1). Controls were used routinely in these assays: positive (assay of supernatant following immunoprecipitation; see Fig. 6A, lane 5 and plasmid p21WAF1 DNA; see Fig. 6A, lane 6) and negative (H2O; see Fig. 6A, lane 7). These data provide definitive evidence that HuR and CP1 interact closely with p21WAF1 mRNA in MDA-468 cells.


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Fig. 6.   HuR and CP1 associate with p21WAF1 mRNA in MDA-468 cells. A, MDA-468 cytoplasmic extract (200 µg) was incubated with 10 µg of HuR (lane 2), CP1 (lane 3), or EGFR (lane 4) antibody, and the resultant immune complexes were precipitated using protein A and G beads. Co-purifying RNA was isolated following a series of washes and analyzed by RT-PCR using p21WAF1-specific primers (see "Materials and Methods"). MDA-468 RNA from an immunoprecipitation supernatant (lane 5) and p21WAF1 plasmid DNA (lane 6) were used as positive controls. Immunoprecipitation with beads alone (no antibody) (lane 1) and water (lane 7) were included as negative controls. p21WAF1-specific product (105 bp) is indicated with an arrow relative to DNA markers (lane 8). B, UVXL as in Fig. 5 with MDA-468 cell extract and WAF1-HuD (lanes 2-5) or WAF1-1/6 (lanes 7-10) riboprobes, followed by immunoprecipitation with anti-HuR mAb (lanes 3 and 8), anti-GST mAb (lanes 4 and 9), and with beads alone (lanes 5 and 10) prior to 8.5% SDS-PAGE and PhosphorImager analysis compared with 14C molecular mass markers (lanes 1 and 6). C, REMSA-supershift assay with cleaved recombinant HuR protein and WAF1-HuD (lanes 1 and 2) or c-fos HuD (lanes 3 and 4) riboprobes. HuR antibody was used to shift RPCs (lanes 2 and 4). UVXL assay (lanes 6-12) using WAF1-1/6 riboprobe and recombinant GST (lane 7) or cleaved CP1 protein (lanes 8-12), in the presence of either poly(C) (lanes 9 and 11) or poly(A) (lanes 10 and 12) is shown compared with 14C molecular mass markers (lane 5). D, UVXL assay with either WAF1-HuD or WAF1-1/6 riboprobes and control (lanes 2 and 8), EGF (lanes 4 and 9)-, or UVC (lanes 5 and 10)-treated MDA-468 cell extracts compared with 14C molecular mass markers (lanes 3 and 6).

To definitively map the binding site of HuR to WAF1-HuD we performed a UVXL assay using MDA-468 extracts and immunoprecipitated the resultant RPCs with HuR antibody. We identified a single major RPC with a molecular mass of ~36 kDa that was not present when the RPCs were precipitated with GST antibody or with beads alone (Fig. 6B, lanes 3-5). Similar results were obtained with the WAF1-1/6 probe (Fig. 6B, lanes 8-10). These findings provided strong evidence that the 36-kDa RPC detected in UVXL in Fig. 5C represents HuR bound to the WAF1-HuD and WAF1-1/6 probes. When tested, thrombin-cleaved recombinant GST-HuR bound to the WAF1-HuD and c-fos HuD probes (Fig. 6C, lanes 1 and 3), and in each case, the RPC could be supershifted with HuR antibody (Fig. 6C, lanes 2 and 4).

To investigate the binding of CP1 to the WAF1-1/6 element, we performed a UVXL assay with thrombin-cleaved recombinant GST-CP1. CP1 protein bound to the WAF1-1/6 riboprobe (Fig. 6C, lane 8); binding could be displaced using poly(C) ribohomopolymer (Fig. 6C, lanes 9 and 11) but not using poly(A) ribohomopolymer (Fig. 6C, lanes 10 and 12), confirming the specificity of this protein species for C-rich sequences. These data suggest that CP1 may target one or more motifs within the UVC-responsive WAF1-1/6 element.

We next used MDA-468 whole cell extracts treated with EGF or UVC in UVXL experiments with WAF1-HuD and WAF1-1/6 probes. In each case, UVC up-regulated binding of HuR (~36 kDa) to the probe, whereas EGF did not (WAF1-HuD; see Fig. 6D, lanes 1-5 and WAF1-1/6; see Fig. 6D, lanes 6-10). A similar UVC-induced increase in binding of HuR to p21WAF1 mRNA has been observed in RKO colorectal carcinoma cells (17). However, no significant change was seen in the pattern of binding for any of the other p21WAF1 RNA-binding proteins. These data suggest an important role for HuR in the UVC-induced up-regulation of p21WAF1 mRNA stability and implicate a lesser role for HuR in the EGF-mediated changes in p21WAF1 mRNA turnover. These results support our transfection data (Fig. 4, C and D) in which EGF up-regulated luciferase reporter activity through sequences downstream of WAF1-HuD and WAF-1/6, whereas UVC regulated luciferase activity primarily through sequences within WAF1-1/6, presumably those which bind HuR.

To determine the functional role of HuR in the regulation of p21WAF1 expression and control of cell cycle in MDA-468 cells, we used retroviral vectors to generate stable pools of MDA-468 cells expressing antisense or sense HuR. HuR protein levels varied significantly between the antisense and sense MDA-468 sublines. Despite this, no significant difference was observed in either p21WAF1 or actin protein levels following EGF treatment (Fig. 7A). We also used flow cytometry to examine the effect of HuR levels on the cell cycle profile. However, no difference was seen between the sublines (Fig. 7B), where the proportion of cells in S phase was unaffected by increasing (sense) or decreasing (antisense) HuR levels. The EGF-induced reduction in S phase content was also unaffected by HuR levels (Fig. 7B). Similarly, in a colony formation assay (not shown), EGF induced >98% reduction in the number of colonies with each subline. Taken together, these data suggest that although HuR binds to the WAF1-HuD sequence of p21WAF1 mRNA (within the context of the larger WAF1-1/6 region) in MDA-468 cells, modification of cellular HuR levels has little or no effect on the regulation of p21WAF1 protein levels or progression of cells through the cell cycle.


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Fig. 7.   HuR does not regulate EGF-induced p21WAF1 expression or cell cycle profile in MDA-468 cells. A, Western blot analysis of HuR, p21WAF1, and actin protein levels in pools of retrovirally infected MDA-468 cells that stably express sense (lane 2) or antisense (lane 3) HuR and were treated for 4 h with EGF (25 ng/ml). Analysis of parental MDA-468 cells is included for comparison (lane 1). AS, antisense. B, MDA-468 sublines indicated in A, as well as vector control-infected cells (Puro), were incubated ± EGF (25 ng/ml) for 16 h and then subjected to flow cytometry analysis (as described under "Materials and Methods") to determine % of cells in S phase.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

p21WAF1 plays a central role in various models of growth inhibition, although the molecular mechanisms that regulate p21WAF1 expression in breast cancer cells are not well understood. Here we have shown that EGF-induced growth inhibition in MDA-468 breast cancer cells (mutant p53) is associated with a rapid increase in p21WAF1 protein and mRNA expression, which results from the combination of increased transcription and stabilization of p21WAF1 mRNA. Significantly, we established that the 3'-UTR of p21WAF1 contains cis-acting elements that modulate the stability of a heterologous reporter mRNA. Through transfection studies, distinct regions have been defined within the p21WAF1 3'-UTR that basally regulate, and confer UVC- and EGF-inducible changes to, heterologous reporter activity. Furthermore, these cis-elements are the target for several RNA-binding proteins, including HuR and CP1. Despite its documented role in mediating the stabilization of a number of transcripts, including p21WAF1 mRNA in UVC-treated RKO colorectal carcinoma cells (17), and in promoting cyclooxygenase-2 expression in colon carcinoma cells (59), our binding assays, together with sense and antisense expression studies, suggest that HuR does not play a major role in the EGF-induced expression of p21WAF1 in MDA-468 breast cancer cells.

The data presented herein support the notion that post-transcriptional pathways are a major regulator of p21WAF1 gene expression. Several compounds have been shown to increase the stability of p21WAF1 mRNA in a variety of cells. These include phorbol ester (phorbol 12-myristate 13-acetate; ~8-fold increase in p21WAF1 mRNA stability in human ovarian carcinoma SKOV-3 cells) (12), tumor necrosis factor alpha  (~5-fold increase in human myeloid leukemic KG-1 cells) (13), a novel retinoid CD437 (~3-fold increase in MDA-468 and MCF-7 breast cancer cells) (14), UV light (~4-fold increase in mouse embryonal fibroblasts) (15), phenylephrine (~3-fold increase in transfected HepG2 cells, mediated by p42/44 MAP kinase) (48), and of direct relevance to this work, EGF (~2-fold in A431 human epidermoid carcinoma cells) (16). This response to EGF may only be apparent in cells that overexpress EGFRs, such as MDA-468 cells and A431 cells, as EGF did not modulate p21WAF1 mRNA stability in MCF-7 cells, which express lower levels of EGFR (16). In each of these reports, where measured, the transcriptional increase in p21WAF1 contributed less than the increase in mRNA stability to the total mRNA level.

Our data provides definitive mRNA decay evidence implicating the p21WAF1 3'-UTR in cis, supporting the findings of Liu et al. (48). Previous studies into the effect of shorter elements, such as WAF1-HuD or WAF1-1/6, demonstrated that the WAF1-HuD region did not destabilize a CAT reporter RNA in transfected HepG2 cells significantly (48). Similarly, Li et al. (48) found that the ARE motifs present within the p21WAF1 3'-UTR (HuD; A, B, and C in Fig. 1, A and B) did not contribute substantially toward message instability in breast cancer cells (14). Instead, they found that the predominant basal instability sequences were contained downstream of the WAF1-1/6 region thought responsible for UVC-induced stabilization of p21WAF1 mRNA. Together with our data, these findings suggest that the p21WAF1 3'-UTR is a composite cis-acting sequence, with contributions to basal turnover from each of the WAF1-1/6, WAF1-879, and WAF1-1512 regions, but that the majority of the effect is because of sequences downstream of WAF1-1/6 (cf. with Li et al. (14)). Interestingly, EGF and UVC augmented reporter activity preferentially via different components of the 3'-UTR. In particular, EGF-induced up-regulation of reporter activity occurred predominantly via a combination of WAF1-879 and WAF1-1512, with little effect via WAF1-1/6. In contrast, UVC augmented reporter activity predominantly through WAF1-1/6, consistent with the findings of Wang et al. (17), together with smaller, yet significant, up-regulation via WAF1-879 and WAF1-1512 sequences. Further analysis of each of these three components of the 3'-UTR will be required in different cell types and with different stimuli to develop a definitive understanding of the mechanisms underlying p21WAF1 mRNA turnover.

We have produced several lines of evidence in support of the association of HuR and CP1 with p21WAF1 mRNA in MDA-468 breast cancer cells. These include the immunoprecipitation of endogenous HuR bound to the WAF1-HuD and WAF1-1/6 riboprobes and the immunoco-purification of p21WAF1 mRNA from MDA-468 cell extracts using HuR and CP1 antibodies. These assays (UVXL-IP and IP-RT-PCR) provide the first definitive evidence for a close association of HuR and CP1 with p21WAF1 mRNA in MDA-468 cells.

Based on these observations and the findings of others, we presumed there would be a significant role for HuR in the EGF-induced regulation of p21WAF1 mRNA stability in breast cancer cells. For example, HuR and other members of the ELAV protein family have been shown to stabilize AU-rich mRNAs in several other cell systems (35). These include the stabilization of VEGF mRNA (36), GLUT-1 mRNA (49), and p21WAF1 mRNA in UVC-treated colorectal carcinoma cells (17). In the latter report, the shortest riboprobe that the authors used was equivalent to our WAF1-1/6 probe (see Fig. 1A). HuR was one of only two proteins detected by UVXL, and HuR antibody produced a partial supershift in cell extracts. Furthermore, antisense HuR-expressing clones (with a 4-6-fold reduction in HuR levels) demonstrated a decrease in both basal and UV-induced p21WAF1 expression (mRNA stability and protein levels). However, we observed that the WAF1-1/6 region did not have a major role in modulating EGF-induced reporter activity in MDA-468 cells. Moreover, we found that treatment of MDA-468 cells with UVC, but not EGF, regulated the binding of HuR to the WAF1-HuD and WAF1-1/6 riboprobes. Thus, we were not surprised to find that modification of HuR expression in MDA-468 cells had no detectable effect on EGF-induced p21WAF1 protein levels, cell cycle, or growth. Taken together, these observations suggest that in these cells, HuR plays a relatively minor role in the regulation of basal and EGF-induced expression of p21WAF1.

In this context, Liu et al. (48) found that non-HuR RNA-binding proteins (24 and 52 kDa) mediated the induction of p21WAF1 mRNA stability by the alpha 1 adrenergic agonist, phenylephrine (48). They did not observe binding of cellular HuR to their riboprobe, which was identical to our WAF1-HuD riboprobe. We therefore presume that the role of HuR in regulating p21WAF1 expression varies according to the mode of stimulus and is dependent upon cell type. It also emphasizes that RNA-binding proteins other than HuR can regulate p21WAF1 mRNA turnover.

The poly(C)-binding proteins, CP1 and CP2, are members of the hnRNP K-homology domain family of RNA-binding proteins (50) and regulate the stability of a variety of transcripts, including alpha -globin, tyrosine hydroxylase, and erythropoeitin (51-55), as well as regulating translation of 15-lipoxygenase and human papillomavirus (56, 57). Co-immunopurification of p21WAF1 mRNA from MDA-468 cells using CP1 antibody suggests that CP1 protein binds to one or more of the motifs distributed throughout the p21WAF1 3'-UTR (see Fig. 1A). UVXL analysis of WAF1-1/6, WAF1-879, and WAF1-1512 demonstrates the presence of RPCs at ~42 kDa, which may contain CP1 and/or CP2. CP1 may therefore play a role in the regulation of p21WAF1 mRNA stability in MDA-468 cells through interactions with sequences within and/or downstream of WAF1-1/6.

We have identified a U- and C-rich cis-element in the 3'-UTR of the human androgen receptor mRNA that is the target for simultaneous, co-operative binding of HuR and CP1/CP2 (47). The UVXL assays presented herein with recombinant CP1 suggest that CP1 may bind to the UVC-responsive WAF1-1/6 element. The close proximity of HuR and CP1 binding sites within WAF1-1/6 might allow both proteins to participate in coordinated mRNA decay. It also emphasizes the need to examine the functional role of CP1 in p21WAF1 mRNA turnover in MDA-468 cells.

In summary, EGF increases p21WAF1 mRNA expression in p53 mutant breast cancer cells through a combination of mRNA stabilization and transcriptional up-regulation. We have identified cis-elements within the p21WAF1 3'-UTR that are distinctly EGF- or UVC-inducible in MDA-468 cells. This implies that different stimuli can regulate p21WAF1 mRNA stability via independent cis-elements. HuR binding modulates p21WAF1 expression in UVC-treated RKO cells but not in EGF-treated MDA-468 cells. This indicates that there is an HuR-independent, cell type-specific mechanism through which EGF induces p21WAF1 expression via stabilization of p21WAF1 mRNA. CP1 and other RNA-binding proteins associate with p21WAF1 mRNA in MDA-468 cells and may direct its turnover. The cloning and characterization of these proteins are the subject of further investigation.

    ACKNOWLEDGEMENTS

We thank Bert Vogelstein for the p21WAF1 cDNA plasmid, Maria Czyzyk-Krzeska for the CP1 antibody, Mike Kiledjian for the GST-CP1 construct, and Robert Medcalf for advice on the HuR supershift assay. We are also grateful to Romano Krueger for assistance with the cell cycle analysis and Britt Granath for advice with the statistical analysis.

    FOOTNOTES

* This work was funded in part by the Raine Medical Research Foundation, the Kathleen Cuningham Foundation for Breast Cancer Research, the Medical Research Foundation of Royal Perth Hospital, and the Cancer Foundation of Western Australia.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by a Faculty of Dentistry and Medicine scholarship from the University of Western Australia.

Supported by Raine Medical Research Foundation fellowship.

Dagger Dagger To whom correspondence should be addressed: University Department of Medicine, Royal Perth Hospital, GPO Box X2213, Perth, Western Australia 6001, Australia. Tel.: 61-8-9224-0323; Fax: 61-8-9224-0246; E-mail: peterl@cyllene.uwa.edu.au.

Published, JBC Papers in Press, November 12, 2002, DOI 10.1074/jbc.M208439200

    ABBREVIATIONS

The abbreviations used are: EGF, epidermal growth factor; EGFR, EGF receptor; UVC, short wavelength ultraviolet light; ActD, actinomycin D; ARE, AU-rich element; CP, poly(C)-binding protein; DTT, dithiothreitol; ELAV, embryonic lethal abnormal vision; GST, glutathione S-transferase; REMSA, RNA electrophoretic mobility shift assay; RPC, RNA-protein complex; UTR, untranslated region; UVXL, UV cross-linking; nt, nucleotide; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1, 3-diol; MES, 4-morpholineethanesulfonic acid; RT, reverse transcriptase; IP, immunoprecipitation.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. El-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Parsons, R., Trent, J. M., Lin, D., Mercer, W. E., Kinzler, K. W., and Vogelstein, B. (1993) Cell 75, 817-825[Medline] [Order article via Infotrieve]
2. Del Sal, G., Murphy, M., Ruaro, E., Lazarevic, D., Levine, A. J., and Schneider, C. (1996) Oncogene 12, 177-185[Medline] [Order article via Infotrieve]
3. Hengst, L., Dulic, V., Slingerland, J. M., Lees, E., and Reed, S. I. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5291-5295[Abstract]
4. Sheikh, M. S., Rochefort, H., and Garcia, M. (1995) Oncogene 11, 1899-1905[Medline] [Order article via Infotrieve]
5. Nigro, J. M., Baker, S. J., Preisinger, A. C., Jessup, J. M., Hostetter, R., Cleary, K., Bigner, S. H., Davidson, N., Baylin, S., Devilee, P., et al.. (1989) Nature 342, 705-708[CrossRef][Medline] [Order article via Infotrieve]
6. Davidson, N. E., Gelmann, E. P., Lippman, M. E., and Dickson, R. B. (1987) Mol. Endocrinol. 1, 216-223[Abstract]
7. Filmus, J., Pollak, M. N., Cailleau, R., and Buick, R. N. (1985) Biochem. Biophys. Res. Commun. 128, 898-905[Medline] [Order article via Infotrieve]
8. Xie, W., Su, K., Wang, D., Paterson, A. J., and Kudlow, J. E. (1997) Anticancer Res. 17, 2627-2633[Medline] [Order article via Infotrieve]
9. Fan, Z., Lu, Y., Wu, X., DeBlasio, A., Koff, A., and Mendelsohn, J. (1995) J. Cell Biol. 131, 235-242[Abstract]
10. Jakus, J., and Yeudall, W. A. (1996) Oncogene 12, 2369-2376[Medline] [Order article via Infotrieve]
11. Daly, J. M., Olayioye, M. A., Wong, A. M., Neve, R., Lane, H. A., Maurer, F. G., and Hynes, N. E. (1999) Oncogene 18, 3440-3451[CrossRef][Medline] [Order article via Infotrieve]
12. Akashi, M., Osawa, Y., Koeffler, H. P., and Hachiya, M. (1999) Biochem. J. 337, 607-616[CrossRef][Medline] [Order article via Infotrieve]
13. Shiohara, M., Akashi, M., Gombart, A. F., Yang, R., and Koeffler, H. P. (1996) J. Cell. Physiol. 166, 568-576[CrossRef][Medline] [Order article via Infotrieve]
14. Li, X. S., Rishi, A. K., Shao, Z. M., Dawson, M. I., Jong, L., Shroot, B., Reichert, U., Ordonez, J., and Fontana, J. A. (1996) Cancer Res. 56, 5055-5062[Abstract]
15. Gorospe, M., Wang, X., and Holbrook, N. J. (1998) Mol. Cell. Biol. 18, 1400-1407[Abstract/Free Full Text]
16. Johannessen, L. E., Knardal, S. L., and Madshus, I. H. (1999) Biochem. J. 337, 599-606[CrossRef][Medline] [Order article via Infotrieve], (Pt 3)
17. Wang, W., Furneaux, H., Cheng, H., Caldwell, M. C., Hutter, D., Liu, Y., Holbrook, N., and Gorospe, M. (2000) Mol. Cell. Biol. 20, 760-769[Abstract/Free Full Text]
18. Hollams, E. H., Giles, K. M., Thomson, A. M., and Leedman, P. J. (2002) Neurochem. Res. 27, 957-980[CrossRef][Medline] [Order article via Infotrieve]
19. Chen, C. Y., and Shyu, A. B. (1994) Mol. Cell. Biol. 14, 8471-8482[Abstract]
20. Shaw, G., and Kamen, R. (1986) Cell 46, 659-667[Medline] [Order article via Infotrieve]
21. Lagnado, C. A., Brown, C. Y., and Goodall, G. J. (1994) Mol. Cell. Biol. 14, 7984-7995[Abstract]
22. Zubiaga, A. M., Belasco, J. G., and Greenberg, M. E. (1995) Mol. Cell. Biol. 15, 2219-2230[Abstract]
23. Makeyev, A. V., and Liebhaber, S. A. (2002) RNA (N. Y.) 8, 265-278
24. Gillis, P., and Malter, J. S. (1991) J. Biol. Chem. 266, 3172-3177[Abstract/Free Full Text]
25. Brewer, G. (1991) Mol. Cell. Biol. 11, 2460-2466[Medline] [Order article via Infotrieve]
26. Levine, T. D., Gao, F., King, P. H., Andrews, L. G., and Keene, J. D. (1993) Mol. Cell. Biol. 13, 3494-3504[Abstract]
27. Hamilton, B. J., Nagy, E., Malter, J. S., Arrick, B. A., and Rigby, W. F. (1993) J. Biol. Chem. 268, 8881-8887[Abstract/Free Full Text]
28. Nakagawa, J., Waldner, H., Meyer-Monard, S., Hofsteenge, J., Jeno, P., and Moroni, C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2051-2055[Abstract]
29. Ma, W. J., Cheng, S., Campbell, C., Wright, A., and Furneaux, H. (1996) J. Biol. Chem. 271, 8144-8151[Abstract/Free Full Text]
30. Chung, S., Jiang, L., Cheng, S., and Furneaux, H. (1996) J. Biol. Chem. 271, 11518-11524[Abstract/Free Full Text]
31. Carballo, E., Lai, W. S., and Blackshear, P. J. (1998) Science 281, 1001-1005[Abstract/Free Full Text]
32. Afonina, E., Neumann, M., and Pavlakis, G. N. (1997) J. Biol. Chem. 272, 2307-2311[Abstract/Free Full Text]
33. Keene, J. D. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 5-7[Free Full Text]
34. Fan, X. C., and Steitz, J. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 15293-15298[Abstract/Free Full Text]
35. Fan, X. C., and Steitz, J. A. (1998) EMBO J. 17, 3448-3460[Abstract/Free Full Text]
36. Levy, N. S., Chung, S., Furneaux, H., and Levy, A. P. (1998) J. Biol. Chem. 273, 6417-6423[Abstract/Free Full Text]
37. Sokolowski, M., Furneaux, H., and Schwartz, S. (1999) J. Virol. 73, 1080-1091[Abstract/Free Full Text]
38. Myer, V. E., Fan, X. C., and Steitz, J. A. (1997) EMBO J. 16, 2130-2139[Abstract/Free Full Text]
39. Joseph, B., Orlian, M., and Furneaux, H. (1998) J. Biol. Chem. 273, 20511-20516[Abstract/Free Full Text]
40. Pear, W. S., Nolan, G. P., Scott, M. L., and Baltimore, D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8392-8396[Abstract/Free Full Text]
41. Thomson, A. M., Rogers, J. T., Walker, C. E., Staton, J. M., and Leedman, P. J. (1999) Biotechniques 27, 1032-1042[Medline] [Order article via Infotrieve]
42. Morgenstern, J. P., and Land, H. (1990) Nucleic Acids Res. 18, 3587-3596[Abstract]
43. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159[CrossRef][Medline] [Order article via Infotrieve]
44. Bjorge, J. D., Paterson, A. J., and Kudlow, J. E. (1989) J. Biol. Chem. 264, 4021-4027[Abstract/Free Full Text]
45. Yeap, B. B., Krueger, R. G., and Leedman, P. J. (1999) Endocrinology 140, 3282-3291[Abstract/Free Full Text]
46. Maurer, F., Tierney, M., and Medcalf, R. L. (1999) Nucleic Acids Res. 27, 1664-1673[Abstract/Free Full Text]
47. Yeap, B. B., Voon, D. C., Vivian, J. P., McCulloch, R. K., Thomson, A. M., Giles, K. M., Czyzyk-Krzeska, M. F., Furneaux, H., Wilce, M. C., Wilce, J. A., and Leedman, P. J. (2002) J. Biol. Chem. 277, 27183-27192[Abstract/Free Full Text]
48. Liu, J., Shen, X., Nguyen, V. A., Kunos, G., and Gao, B. (2000) J. Biol. Chem. 275, 11846-11851[Abstract/Free Full Text]
49. Jain, R. G., Andrews, L. G., McGowan, K. M., Pekala, P. H., and Keene, J. D. (1997) Mol. Cell. Biol. 17, 954-962[Abstract]
50. Kiledjian, M., Wang, X., and Liebhaber, S. A. (1995) EMBO J. 14, 4357-4364[Abstract]
51. Wang, X., Kiledjian, M., Weiss, I. M., and Liebhaber, S. A. (1995) Mol. Cell. Biol. 15, 1769-1777[Abstract]
52. Paulding, W. R., and Czyzyk-Krzeska, M. F. (1999) J. Biol. Chem. 274, 2532-2538[Abstract/Free Full Text]
53. Czyzyk-Krzeska, M. F., and Beresh, J. E. (1996) J. Biol. Chem. 271, 3293-3299[Abstract/Free Full Text]
54. McGary, E. C., Rondon, I. J., and Beckman, B. S. (1997) J. Biol. Chem. 272, 8628-8634[Abstract/Free Full Text]
55. Czyzyk-Krzeska, M. F., and Bendixen, A. C. (1999) Blood 93, 2111-2120[Abstract/Free Full Text]
56. Ostareck, D. H., Ostareck-Lederer, A., Wilm, M., Thiele, B. J., Mann, M., and Hentze, M. W. (1997) Cell 89, 597-606[Medline] [Order article via Infotrieve]
57. Collier, B., Goobar-Larsson, L., Sokolowski, M., and Schwartz, S. (1998) J. Biol. Chem. 273, 22648-22656[Abstract/Free Full Text]
58. Daly, J. M., Jannot, C. B., Beerli, R. R., Graus-Porta, D., Maurer, F. G., and Hynes, N. E. (1997) Cancer Res. 57, 3804-3811[Abstract]
59. Dixon, D. A., Tolley, N. D., King, P. H., Nabors, L. H., McIntyre, T. M., Zimmerman, G. A., and Prescott, S. M. (2001) J. Clin. Invest. 108, 1657-1665[Abstract/Free Full Text]
60. Kiledjian, M., Day, N., and Trifillis, P. (1999) Methods 17, 84-91[CrossRef][Medline] [Order article via Infotrieve]


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