Ets2 Maintains hTERT Gene Expression and Breast Cancer Cell Proliferation by Interacting with c-Myc
2008; Elsevier BV; Volume: 283; Issue: 35 Linguagem: Inglês
10.1074/jbc.m800790200
ISSN1083-351X
AutoresDakang Xu, Julie Dwyer, Li He, Wei Duan, Jun‐Ping Liu,
Tópico(s)Genomics and Chromatin Dynamics
ResumoHuman telomerase reverse transcriptase (hTERT) underlies cancer cell immortalization, and the expression of hTERT is regulated strictly at the gene transcription. Here, we report that transcription factor Ets2 is required for hTERT gene expression and breast cancer cell proliferation. Silencing Ets2 induces a decrease of hTERT gene expression and increase in human breast cancer cell death. Reconstitution with recombinant hTERT rescues the apoptosis induced by Ets2 depression. In vitro and in vivo analyses show that Ets2 binds to the EtsA and EtsB DNA motifs on the hTERT gene promoter. Mutation of either Ets2 binding site reduces the hTERT promoter transcriptional activity. Moreover, Ets2 forms a complex with c-Myc as demonstrated by co-immunoprecipitation and glutathione S-transferase pulldown assays. Immunological depletion of Ets2, or mutation of the EtsA DNA motif, disables c-Myc binding to the E-box, whereas removal of c-Myc or mutation of the E-box also compromises Ets2 binding to EtsA. Thus, hTERT gene expression is maintained by a mechanism involving Ets2 interactions with the c-Myc transcription factor and the hTERT gene promoter, a protein-DNA complex critical for hTERT gene expression and breast cancer cell proliferation. Human telomerase reverse transcriptase (hTERT) underlies cancer cell immortalization, and the expression of hTERT is regulated strictly at the gene transcription. Here, we report that transcription factor Ets2 is required for hTERT gene expression and breast cancer cell proliferation. Silencing Ets2 induces a decrease of hTERT gene expression and increase in human breast cancer cell death. Reconstitution with recombinant hTERT rescues the apoptosis induced by Ets2 depression. In vitro and in vivo analyses show that Ets2 binds to the EtsA and EtsB DNA motifs on the hTERT gene promoter. Mutation of either Ets2 binding site reduces the hTERT promoter transcriptional activity. Moreover, Ets2 forms a complex with c-Myc as demonstrated by co-immunoprecipitation and glutathione S-transferase pulldown assays. Immunological depletion of Ets2, or mutation of the EtsA DNA motif, disables c-Myc binding to the E-box, whereas removal of c-Myc or mutation of the E-box also compromises Ets2 binding to EtsA. Thus, hTERT gene expression is maintained by a mechanism involving Ets2 interactions with the c-Myc transcription factor and the hTERT gene promoter, a protein-DNA complex critical for hTERT gene expression and breast cancer cell proliferation. The Ets protein family consists of a large number of evolutionarily conserved gene transcription factors. Each Ets member has an 85-amino acid sequence called the Ets domain that adopts the winged helix-turn-helix DNA binding structure with high affinity for the core consensus sequence of GGAA/T in a variety of genes. Although many Ets proteins are transcription factors, some function as repressors, and others are shown to be both activators and repressors (1Seth A. Watson D.K. Eur. J. Cancer. 2005; 41: 2462-2478Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar, 2Mavrothalassitis G. Ghysdael J. Oncogene. 2000; 19: 6524-6532Crossref PubMed Scopus (111) Google Scholar, 3Myers E. Hill A.D. Kelly G. McDermott E.W. O'Higgins N.J. Buggy Y. Young L.S. Clin. Cancer Res. 2005; 11: 2111-2122Crossref PubMed Scopus (102) Google Scholar, 4Li R. Pei H. Watson D.K. Oncogene. 2000; 19: 6514-6523Crossref PubMed Scopus (188) Google Scholar). Frequently, Ets1 and Ets2 are highly expressed in a number of human malignancies (1Seth A. Watson D.K. Eur. J. Cancer. 2005; 41: 2462-2478Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar, 3Myers E. Hill A.D. Kelly G. McDermott E.W. O'Higgins N.J. Buggy Y. Young L.S. Clin. Cancer Res. 2005; 11: 2111-2122Crossref PubMed Scopus (102) Google Scholar, 5Hsu T. Trojanowska M. Watson D.K. J. Cell Biochem. 2004; 91: 896-903Crossref PubMed Scopus (222) Google Scholar, 6Galang C.K. Muller W.J. Foos G. Oshima R.G. Hauser C.A. J. Biol. Chem. 2004; 279: 11281-11292Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Overexpression of Ets1 and Ets2 stimulates cell proliferation, anchorage-independent growth, and tumorigenicity in nude mice (7Hahne J.C. Okuducu A.F. Kaminski A. Florin A. Soncin F. Wernert N. Oncogene. 2005; 24: 5384-5388Crossref PubMed Scopus (61) Google Scholar, 8Foos G. Hauser C.A. Oncogene. 2000; 19: 5507-5516Crossref PubMed Scopus (32) Google Scholar). Targeted disruption of a single allele of the Ets2 gene limits the growth of breast tumors in transgenic mice (9Neznanov N. Man A.K. Yamamoto H. Hauser C.A. Cardiff R.D. Oshima R.G. Cancer Res. 1999; 59: 4242-4246PubMed Google Scholar), whereas homozygote deletion is embryonic lethal (10Yamamoto H. Flannery M.L. Kupriyanov S. Pearce J. McKercher S.R. Henkel G.W. Maki R.A. Werb Z. Oshima R.G. Genes Dev. 1998; 12: 1315-1326Crossref PubMed Scopus (251) Google Scholar). These findings suggest that Ets2 plays an important role in the development of breast cancer.The molecular mechanisms of action of Ets2 in tumorigenesis remain largely unknown. Ets2 is phosphorylated on threonine 72 by ERK1/2 and the phosphorylated form accumulates in the cell nucleus (11Yang B.S. Hauser C.A. Henkel G. Colman M.S. Van Beveren C. Stacey K.J. Hume D.A. Maki R.A. Ostrowski M.C. Mol. Cell. Biol. 1996; 16: 538-547Crossref PubMed Scopus (316) Google Scholar). Stimulation of VEGFR2, ErbB2, or estrogen receptors results in activation of ERK1/2 5The abbreviations used are:ERKextracellular signal-regulated kinaseTERTtelomerase reverse transcriptaseChIPchromatin immunoprecipitationPTHrPparathyroid hormone-related proteinRNAiRNA interferenceHAhemagglutininsiRNAsmall interfering RNARTreverse transcriptasePBSphosphate-buffered salineGSTglutathione S-transferaseFACSfluorescence-activated cell sortingGFPgreen fluorescent proteinCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. 5The abbreviations used are:ERKextracellular signal-regulated kinaseTERTtelomerase reverse transcriptaseChIPchromatin immunoprecipitationPTHrPparathyroid hormone-related proteinRNAiRNA interferenceHAhemagglutininsiRNAsmall interfering RNARTreverse transcriptasePBSphosphate-buffered salineGSTglutathione S-transferaseFACSfluorescence-activated cell sortingGFPgreen fluorescent proteinCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. kinases and phosphorylation of Ets2 in human breast cancer cells (12Svensson S. Jirstrom K. Ryden L. Roos G. Emdin S. Ostrowski M.C. Landberg G. Oncogene. 2005; 24: 4370-4379Crossref PubMed Scopus (100) Google Scholar, 13Man A.K. Young L.J. Tynan J.A. Lesperance J. Egeblad M. Werb Z. Hauser C.A. Muller W.J. Cardiff R.D. Oshima R.G. Mol. Cell. Biol. 2003; 23: 8614-8625Crossref PubMed Scopus (54) Google Scholar). Several downstream target genes of Ets2 have been implicated as mediating Ets2-induced oncogenesis. These include Bcl-2, cyclin D1, and Bcl-XL in prostate cancer (14Carbone G.M. Napoli S. Valentini A. Cavalli F. Watson D.K. Catapano C.V. Nucleic Acids Res. 2004; 32: 4358-4367Crossref PubMed Scopus (71) Google Scholar, 15Sementchenko V.I. Schweinfest C.W. Papas T.S. Watson D.K. Oncogene. 1998; 17: 2883-2888Crossref PubMed Scopus (65) Google Scholar). Recently, telomerase reverse transcriptase (hTERT) has been suggested to be a target gene of the Ets family (reviewed Ref. 16Dwyer J. Li H. Xu D. Liu J.P. Ann. N. Y. Acad. Sci. 2007; 1114: 36-47Crossref PubMed Scopus (69) Google Scholar). However, controversy remains as to whether Ets2 is a transcription factor (17Maida Y. Kyo S. Kanaya T. Wang Z. Yatabe N. Tanaka M. Nakamura M. Ohmichi M. Gotoh N. Murakami S. Inoue M. Oncogene. 2002; 21: 4071-4079Crossref PubMed Scopus (124) Google Scholar) or repressor (18Xiao X. Athanasiou M. Sidorov I.A. Horikawa I. Cremona G. Blair D. Barret J.C. Dimitrov D.S. Exp. Mol. Pathol. 2003; 75: 238-247Crossref PubMed Scopus (30) Google Scholar, 19Xiao X. Phogat S.K. Sidorov I.A. Yang J. Horikawa I. Prieto D. Adelesberger J. Lempicki R. Barrett J.C. Dimitrov D.S. Leukemia. 2002; 16: 1877-1880Crossref PubMed Scopus (17) Google Scholar). Maida et al. showed that mutation of a putative Ets binding site proximal to the transcription site (–23TTCCTT–18) on the hTERT gene promoter inhibits, and overexpression of Ets2 stimulates, the recombinant hTERT gene promoter activity in vulvar cancer A431 cells (17Maida Y. Kyo S. Kanaya T. Wang Z. Yatabe N. Tanaka M. Nakamura M. Ohmichi M. Gotoh N. Murakami S. Inoue M. Oncogene. 2002; 21: 4071-4079Crossref PubMed Scopus (124) Google Scholar). Xiao et al. (18Xiao X. Athanasiou M. Sidorov I.A. Horikawa I. Cremona G. Blair D. Barret J.C. Dimitrov D.S. Exp. Mol. Pathol. 2003; 75: 238-247Crossref PubMed Scopus (30) Google Scholar, 19Xiao X. Phogat S.K. Sidorov I.A. Yang J. Horikawa I. Prieto D. Adelesberger J. Lempicki R. Barrett J.C. Dimitrov D.S. Leukemia. 2002; 16: 1877-1880Crossref PubMed Scopus (17) Google Scholar) showed that both Ets1 and Ets2 bind to the hTERT gene promoter and overexpression of either Ets1 or Ets2 inhibits telomerase activity, an effect independent of gene expression of c-Myc and Sp1 in human myeloid erythroleukemia K562 cells.The regulation of hTERT gene transcription is an important step toward telomerase activation, maintenance of telomeres (ends of chromosomes), and continuous cell proliferation. This is particularly critical in cancer cells where telomeres are critically short in supporting constant cell proliferation. Previous studies show that c-myc is a primary transcription factor of the hTERT gene. Binding the E-box on the hTERT gene promoter, c-myc, stimulates hTERT gene promoter activity (16Dwyer J. Li H. Xu D. Liu J.P. Ann. N. Y. Acad. Sci. 2007; 1114: 36-47Crossref PubMed Scopus (69) Google Scholar, 20Li H. Xu D. Li J. Berndt M.C. Liu J.P. J. Biol. Chem. 2006; 281: 25588-25600Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Myc regulates gene expression by binding cognate DNA sequences known as E-box (CACGTG). Although other transcriptional factors such as Ets1, Ets2, and Sp1 are also implicated as playing a role in regulating the hTERT gene, little is known of and how these transcription factors cooperate.To establish the role of Ets1 and Ets2 in regulating telomerase activity, we chose to silence each of the Ets1 and Ets2 genes in breast cancer cells in which Ets2 is expressed at high levels (6Galang C.K. Muller W.J. Foos G. Oshima R.G. Hauser C.A. J. Biol. Chem. 2004; 279: 11281-11292Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), to observe their effects on hTERT gene expression and breast cancer cell proliferation. We found that knockdown of Ets1 has no effect on telomerase activity, but knockdown of Ets2 significantly disrupts hTERT gene expression and causes breast cancer cell death. Reconstitution with recombinant hTERT rescues the Ets2 knockdown-induced cell death. Mechanistic studies showed that Ets2 binds to the hTERT gene at two discrete Ets-binding sites of the gene promoter. Mutation of either site reduces the hTERT gene promoter activity. In addition, silencing Ets2 entrains a decreased gene expression of c-Myc, but reconstituted c-Myc in the Ets2 knockdown cells does not restore hTERT activity. Furthermore, Ets2 forms a complex with c-Myc regulating c-Myc binding to the E-box. Thus, the hTERT gene is stimulated with a cooperative interaction between Ets2 and c-Myc in their respective bindings to their individual binding sites on the hTERT gene promoter, which is critically required for telomerase activity and hTERT-dependent proliferation of human breast cancer cells.EXPERIMENTAL PROCEDURESCell Culture—The human breast cancer MCF-7 cells were obtained from American Type Culture Collection (Rockville, MD) and grown in Dulbecco's modified Eagle's medium. Cell culture media were supplemented with 10% heat-inactivated fetal calf serum, penicillin (100 units/ml), and streptomycin (100 μg/ml) at 37 °C in a humidified 5% CO2 atmosphere. Cells were seeded in 24-well plastic plates, 6–10-cm dishes or eight-chamber glass slides (Nunc, Naperville, CT).RNA Interference (RNAi) Constructs, Synthetic siRNAs, and Gene Silencing—For generation of Ets1- and Ets2-specific RNAi, specifically annealed oligonucleotides were cloned individually to the BamHI and BbsI sites immediately downstream of the initiating "G" of the U6 promoter. The annealed oligonucleotides used are listed below: Ets2a (5′-tttgtcttgtggatgatgttcttgaagcttgaagaacatcatccacaagactttttt-3′ and 5′-gatcaaaaaagtcttgtggatgatgttcttcaagcttcaagaacatcatccacaaga-3′), Ets2b (5′-tttgcacaggctttaattgtaaagaagcttgtttacaattaaagcctgtgctttttt-3′ and 5′-gatcaaaaaagcacaggctttaattgtaaacaagcttcaagcttgtttacaattaaagcctgtgc-3′), and Ets1 (5′-tttggcagaattcagtgaatcatcggaagcttgcgatgattcactgaattctgctttttt-3′ and 5′-gatcaaaaaagcagaattcagtgaatcatcgcaagcttccgatgattcactgaattctgc-3′). Correctly integrated clones were identified by HindIII digestion and verified by sequencing. The DNA fragment containing the U6 promoter and Ets2-specific hairpin was prepared by amplification with T7 and SP6 and gel purification. 500 ng of U6-Ets2RNAi DNA was co-transfected with 50 ng of a PGK-puromycin into MCF-7 cells using FuGENE 6 (Roche). After 24 h 25 μg/ml puromycin was added to the cultures and after 7 days of selection, cells were pooled and expanded for analysis. For transient gene silencing, synthetic small interference RNA (siRNA) targeting Ets1 and Ets2 were used. The siRNAs were purchased from Ambion.Gene Expressions of Ets2, c-Myc, and hTERT—Wild type hTERT gene expression plasmid was prepared by subcloning hTERT full-length cDNA into the pcDNA3HA plasmid as described previously (21Cao Y. Li H. Deb S. Liu J.P. Oncogene. 2002; 21: 3130-3138Crossref PubMed Scopus (196) Google Scholar). The c-Myc gene expression plasmid was generated by inserting full-length c-Myc cDNA downstream of the EF-1α promoter. The gene expression plasmids were transfected into Ets2 knockdown clones (clone 2–15) using FuGENE 6, e.g. 20 μg of pEFBOS-c-Myc (or pEFBOS 1 FLAG) and a puromycin resistance clone. Individual clones were isolated and screened by PCR. Other Ets2 knockdown cell clones (clones 17–34) were also transfected in mass cell cultures. The transfection of hTERT was confirmed by telomerase activity assay and c-Myc expression was confirmed by Western blotting. The cell line and multiple clones displayed various levels of c-Myc protein; however, there was no difference between their growths (data not shown).Real-time RT-PCR Analysis—Single-stranded cDNA was generated using 1 μg of total RNA as template and avian myeloblastosis virus reverse transcriptase (Promega), as per the manufacturer's instructions. A ⅕ dilution of the resulting cDNA was used for real-time RT-PCR of 45 cycles, using a Light Cycler (Roche, version 3.5 software). Fast start SYBR Green Master Mix (Roche) was used, with 10 pmol of each primer in 10-μl reactions. Primers specific for different genes are: hTERT (5′-CCACCTTGACAAAGTACAG-3′ and 5′-CGTCCAGACTCCGCTTCAT-3′) and human glyceraldehyde-3-phosphate dehydrogenase (5′-GAGAGACCCTCACTGCTG-3′ and 5′-GTAGGTATATGACAAGGTG-3′). Following gel electrophoresis on 2% agarose-TAE gel, the size and purity of each PCR product were analyzed. Each DNA sequence was subjected to BLAST analysis to confirm that the PCR represented the correct gene. All samples were subjected to PCR with glyceraldehyde-3-phosphate dehydrogenase as a control. To create a standard curve, five 10-fold dilutions of plasmid DNA were used as template (10–5–10–9 ng/μl). Results represent an average of three independent experiments.Gene Transfection and Luciferase Gene Reporter Assay—The Ets consensus sites (EtsA and -B) were mutated with the QuikChange site-directed mutagenesis kit (Stratagene) in the hTERT luciferase reporter (pTERT-luc330, gift from Silvia Bacchetti and Yu-sheng Cong). The mutagenesis primers used for EtsA were 5′-CCC AGG ACC GCG CTT CTC ACG TGG CGG AGG G-3′ and 5′-CCC TCC GCC ACG TGA GAA GCG CGG TCC TGG G-3, and for EtsB were 5′-CAG CCC CTC CCC TTC TTT TCC GCG GCC CCG-3′ and 5′-CGG GGC CGC GGA AAA GAA GGG GAG GGG CTG-3′ (underlined were putative Ets binding sites and italic were the mutations). All the expression plasmids, wild type and mutants, were verified by DNA sequencing before use. All plasmids used in the transfection assay were prepared with the endotoxin-free plasmid Maxi-kit (Qiagen) and resuspended with endotoxin-free 0.1× Tris/EDTA buffer to a concentration of 1 μg/μl. MCF-7 cells (2 × 105) were placed in 1 ml of medium in 24-well tissue culture plates and incubated overnight. Next day, the wild type or mutant hTERT promoter reporter gene vector (1 μg) along with 0.2 μgof α-galactosidase expression vector were transfected into MCF-7 cells with FuGENE 6 (Roche). In 24 h of transfection, the cells were harvested, washed in PBS, and lysed in cell lysis buffer (Promega). Luciferase activity was measured with luciferase substrate purchased from Promega according to the procedures of the Luciferase Assay System (Promega), and readout in opaque 96-well plates using a plate reading luminometer. β-Galactosidase was used to normalize transfection efficiency. Experiments were performed in triplicate.Electrophoretic Mobility Shift Assay—MCF-7 cell nuclear extract was incubated with 10,000 cpm of a 22-bp DNA oligonucleotide containing the EtsA and -B consensus sequence (wild type EtsA, 5′-GACCGCGCTTCCCACGTGGC-3′, mutant EtsA, 5′-GACCGCGCTTCTCACGTGGC-3′; wild type EtsB, 5′-CCCTCCCCTTCCTTTCCGCG-3′, and mutant EtsB, 5′-CCCTCCCCTTCTTTTCCGCG-3′). Probes were labeled with [γ-32P]dATP (Amersham Biosciences). Incubations were performed at room temperature for 30 min in the presence of 2 μg of poly(dI-dC) as nonspecific competitor and 10 mm Tris-HCl, pH 7.5, containing 100 mm NaCl, 1 mm EDTA, 5 mm dithiothreitol, 4% glycerol, and 100 μg/ml nuclease-free bovine serum albumin. For competition studies, unlabeled wild-type or mutant Ets oligonucleotides were added to the binding reaction 30 min before the addition of the radiolabeled probe. Specific Ets1 or Ets2 antibody (0.5 μl) was incubated with nuclear extracts for 20 min on ice before the binding reaction. All incubation mixtures were subjected to electrophoresis on native 5% (w/v) polyacrylamide gels, which were subsequently dried and subjected to autoradiography.Chromatin Immunoprecipitation (ChIP) Assays—ChIP assays were performed using the ChIP Assay Kit (Upstate, Lake Placid, NY) according to the manufacturer's protocol. Briefly, MCF-7 cells (1 × 107) were fixed with formaldehyde (final concentration, 1%) for 10 min at room temperature. After washing with phosphate-buffered saline, cells were pelleted and resuspended in SDS lysis buffer (1% SDS, 10 mm EDTA, 50 mm Tris-HCl, pH 8.1, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride). The lysates were sonicated to generate the DNA fragments, diluted with dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mm EDTA, 16.7 mm Tris-HCl, pH 8.1, 167 mm NaCl), and pre-cleared by incubation with a salmon sperm DNA, protein A,agarose 50% slurry for 60 min at 4 °C. The supernatant was incubated with anti-Ets1, anti-Ets2, anti-acetylhistone H3 antibodies or IgG at 4 °C overnight. Immunocomplexes were collected with a salmon sperm DNA, protein A, agarose, 50% slurry, heated at 65 °C to reverse cross-linkage, and treated with 40 μg/ml proteinase K at 45 °C for 60 min. DNA was recovered by phenol/chloroform/ethanol precipitation and used as a template for PCR to amplify the proximal region containing the Ets site of the hTERT promoter or the distal region void of the Ets site as control. The primer pairs for the proximal region were 5′-GGC CGG GCT CCC AGT GGA TTC-3′ and 5′-CAG CGG GGA GCG CGC GGC ATC G-3′, and the primer pairs for the distal region were 5′-GGCAGGCACGAGTGATTTTA-3′ and 5′-CTGAGGCACGAGAATTGCTT-3. Primer pairs for PTHrP control were 5′-TGCCTCGAGCGTGTGAACA-3′ and 5-TCCCATAGCAATGTCTAATTAATCTGG-3. The PCR conditions were 30 s at 94 °C, 30 s at 58 °C, and 30 s at 72 °C for 35 cycles. The PCR products were resolved on a 7% polyacrylamide gel and stained with SYBR Gold (Molecular Probes, Eugene, OR).GST Fusion Proteins—GST, GST-Ets1, and GST-Ets2 fusion proteins were produced in BL21 Escherichia coli under induction by isopropyl 1-thio-β-d-galactopyranoside at room temperature. Proteins were purified by affinity absorption using glutathione-Sepharose 4B beads. The recombinant GST, GST-Ets1, and GST-Ets2 proteins on the glutathione beads were incubated with nuclear protein extracts or total cell lysates of breast cancer MCF-7 cells at 4 °C for 1 h followed by extensive washing. Proteins on the beads were resolved on 8% SDS-PAGE and visualized by immunoblotting with anti-myc antibody and anti-GST antibodies.Immunoprecipitation and Western Blotting Analysis—For immunoprecipitation, cells were lysed with triple detergent lysis buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 0.02% sodium azide, 0.1% SDS, 100 μg/ml phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1% Nonidet P-40, 0.5% sodium deoxycholate). Cell lysates (∼1 mg) were incubated with antibodies as indicated in individual experiments. Antibody complexes were captured using protein A-agarose beads (GE Healthcare) and washed three times with PBS. The beads carrying the immune complex were boiled in SDS sample buffer and analyzed by Western blotting. For total nuclear protein extraction, 1 × 106 cells were collected in cold PBS and resuspended in RIPA buffer (150 mm sodium chloride, 50 mm Tris-HCl, pH 7.4, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% SDS, 5 μg/ml aprotinin, 5 μg/ml leupeptin). Protein concentration was determined using a Bio-Rad protein assay. Proteins were separated by 10% SDS-PAGE and transferred onto nitrocellulose membrane by electroblotting. The nitrocellulose membranes were blocked at 4 °C overnight in 10% (w/v) nonfat milk (0.2% fetal calf serum, 0.05% Tween 20 in 1× PBS) and incubated at room temperature for 1 h with antibodies (Santa Cruz Biotechnology), followed by appropriate horseradish peroxidase-conjugated secondary antibody (diluted 1:1000, DAKO Australia). SuperSignal West PICO (Calbiochem) chemiluminescent substrate kit was used to detect and visualize protein antigens after exposure to BioMax autoradiographic film (Kodak). Alternatively x-ray film or Odyssey was used for autoradiography. The autoradiograph films were scanned and the bands quantified on a PhosphorImager (Fujifilm FLA-2000, Berthold).Immunofluorescence—MCF-7 cells (1 × 105) were seeded in Chamber slides (Lab-Tek II, Nalge Nunc International) containing Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum. Cells at 60–80% confluence were washed with PBS, starved by addition of serum-free Dulbecco's modified Eagle's medium for 24 h, followed by fixation with 4% paraformaldehyde in PBS for 10 min and blocking with CAS blocking solution (Zymed Laboratories Inc.) at room temperature for 30 min. The cells were incubated with the Ets2 polyclonal (Santa Cruz) or hTERT primary antibody at room temperature for 1 h, washed three times with PBS, 0.01% Triton X-100 for 5 min each, and incubated at room temperature for 1 h with fluorescein isothiocyanate-conjugated anti-rabbit secondary antibodies (Molecular Probes). For staining of the nuclei, cells were stained with 50 ng/ml Hoechst (Sigma) at room temperature for 10 min. Slides were washed with PBS, Triton X-100, mounted in anti-fade medium (Bio-Rad), and analyzed by fluorescence microscopy (Leica Instruments).Telomerase Activity Assay Analysis—Atelomeric repeat amplification protocol was employed to determine telomerase activity essentially as described previously (22Li H. Zhao L. Yang Z. Funder J.W. Liu J.P. J. Biol. Chem. 1998; 273: 33436-33442Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Briefly, cells treated with different reagents were washed and lysed by detaching and passing the cells though a 26½-gauge needle attached to a 1-ml syringe in pre-chilled telomeric repeat amplification protocol lysis buffer (0.5% CHAPS, 10 mm Tris-HCl, pH 7.5, 1 mm MgCl2, 63 mm KCl, 0.05% Tween 20, 1 mm EDTA, 10% glycerol, 5 mm β-mercaptoethanol and mixture protease inhibitors). The nuclei were isolated by centrifugation and protein content determined. Equal amounts of nuclear telomerase extracts (0.4 μg) were incubated with telomeric DNA substrate and dNTP, and de novo synthesized telomeric DNA, with or without phenol and chloroform extraction, was amplified by PCR using specific telomeric DNA primers in the presence of [α-32P]ATP (Amersham Biosciences) and Taq DNA polymerase. The resultant 32P-labeled telomeric DNA ladders were resolved by polyacrylamide slab gel electrophoresis followed by autoradiography. To monitor nonspecific PCR effects, additional primers were included: NT (ATCGCTTCTCGGCCTTTT) and TSNT (AATCCGTCGAGCAGAGTTAAAAGGCCGAGAAGCGAT). Negative controls that were treated with either RNase A or alkaline phosphatase to inactivate telomerase were included in each experiment.Apoptosis and the Cell Cycle Analysis—Apoptosis and the cell cycle analysis were performed as described (23Gong J. Traganos F. Darzynkiewicz Z. Anal. Biochem. 1994; 218: 314-319Crossref PubMed Scopus (646) Google Scholar). Briefly, both adherent and floating cells were fixed with 80% cold ethanol, pelleted by centrifugation, stained with propidium iodide and analyzed using an EPICS 752 flow cytometer. Cell debris, doublets, and fixation artifacts were gated out, and G0-G1, S, G2-M, and apoptotic populations recorded on a logarithmic scale. Apoptotic cells were identified as the lower fluorescence peak ("sub-G1 peak" on DNA frequency histograms) due to their reduced DNA content or detected with Apoptaq TUNEL kit (Oncor Inc., Gaithersburg, MD). Alternatively, apoptotic cells were stained with Annexin V-FLUOS conjugate (Roche Diagnostics) and propidium iodide (Sigma) at room temperature for 15 min in an incubation buffer that facilitates binding per the manufacturer's instructions. Analyzed for Annexin V and propidium iodide staining in FL-2 and FL-3 channels, respectively, the percentages of stained cells were determined using a FACSCalibur flow cytometer (BD Biosciences). An acquisition gate was set to include ∼20,000 of the centrally located cells for each sample acquisition using linear forward scatter versus linear side scatter. This acquisition strategy resulted in ∼40,000 ungated events being included for each sample analysis. Dot-plot integration was determinate from the background fluorescence using unstained cells. This integration cursor placement remained unchanged when stained cells were analyzed.RESULTSRequirement of Ets2 for Telomerase Activity and hTERT Gene Expression in Breast Cancer Cells—To investigate the role of Ets1 and Ets2 in regulating telomerase, we selectively silenced each of the Ets1 and Ets2 genes using small hairpin RNAs (shRNA) or synthetic siRNA targeting Ets1 and Ets2 mRNA specifically. MCF-7 cells were transfected stably with Ets1 shRNA construct, Ets2 shRNA constructs, or transiently with siRNA for 48 h. Whereas silencing Ets1 had no significant effect on telomerase activity, silencing Ets2 induced a marked inhibition of telomerase activity to 30–40% of the controls (Fig. 1A). Immunofluorescence staining of the telomerase catalytic subunit hTERT showed significant reduction of hTERT in the nucleus of Ets2-silenced cells (Fig. 1B). Consistent with the reduced telomerase activity and hTERT immunoreactivity, hTERT gene expression was decreased by about 75% in Ets2-silenced cells compared with controls expressing GFP or Ets1 shRNA (Fig. 1C). Western blotting confirmed a significant reduction of the Ets1 and Ets2 immunoreactivities induced by Ets1 and Ets2 RNAi, respectively (Fig. 1D). Thus, silencing the Ets2 gene triggered an inhibition of hTERT gene expression and telomerase activity in breast cancer MCF-7 cells.To explore the specificity and mechanisms of hTERT gene repression induced by Ets2 gene silencing, we examined Bcl-2 and Bcl-xL that were shown to be downstream target genes of Ets2 (14Carbone G.M. Napoli S. Valentini A. Cavalli F. Watson D.K. Catapano C.V. Nucleic Acids Res. 2004; 32: 4358-4367Crossref PubMed Scopus (71) Google Scholar). No significant change of the Bcl-2 or Bcl-xL gene expression was observed by Western blotting using specific antibodies in the same experiment where decreased gene expressions of Ets2 and hTERT were observed (Fig. 1D). However, we found that the proto-oncogene c-Myc was reduced significantly (Fig. 1E). The decrease of c-Myc was about 25% of controls, versus ∼70% decrease of the hTERT gene expression (Fig. 1F). The data suggest that hTERT and c-Myc are downstream target genes of Ets2, and that the hTERT gene is regulated by Ets2 directly and indirectly via c-Myc in human breast cancer cells.Ets2 Gene Silencing, hTERT Gene Repression, and Breast Cancer Cell Death—Because little is known of the cellular consequence of Ets regulation of telomerase activity, we determined the effect of gene silencing of Ets2 on breast cancer cell death by measuring Annexin V and propidium iodide staining-positive cells using fluorescence-activated cell sorting (FACS) analysis. As shown in Fig. 2A, 48 h post-transfection of MCF-7 cells with different shRNA plasmids, there was a significant inhibition of cell proliferation in cell cultures transfected with Ets2 shRNA, whereas control cells underwent exponential cell population doublings. The reduction of cell numbers in the Ets2-transfected cells was by about 70–75% of the controls transfected with GFP shRNA (Fig. 2A). Significant floating cells were noted under the microscope and staining of the attached cells with crystal violet blue showed a significant cell loss (not shown). DNA da
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