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Small Inhibitory RNA Duplexes for Sp1 mRNA Block Basal and Estrogen-induced Gene Expression and Cell Cycle Progression in MCF-7 Breast Cancer Cells

Small Inhibitory RNA Duplexes for Sp1 mRNA Block Basal and Estrogen-induced Gene Expression and... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 32, Issue of August 9, pp. 28815–28822, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Small Inhibitory RNA Duplexes for Sp1 mRNA Block Basal and Estrogen-induced Gene Expression and Cell Cycle Progression in MCF-7 Breast Cancer Cells* Received for publication, April 19, 2002, and in revised form, May 24, 2002 Published, JBC Papers in Press, June 6, 2002, DOI 10.1074/jbc.M203828200 Maen Abdelrahim‡, Ismael Samudio‡, Roger Smith III§, Robert Burghardt¶, and Stephen Safe‡ From the Departments of ‡Veterinary Physiology and Pharmacology, §Veterinary Pathobiology, and ¶Anatomy and Public Health, Texas A&M University, College Station, Texas 77843-4466 Small interfering RNA duplexes containing 21–22 nucle- other members of the nuclear receptor family of transcription otides that mediate sequence-specific mRNA degradation factors (11–19), and research in our laboratory has focused on and inhibitory RNA (iRNA) for Sp1 mRNA were used in the molecular mechanisms of the ligand-dependent activation this study to investigate the role of Sp1 on basal and of ER/Sp1 in breast and endometrial cancer cell lines (20 –31). hormone-induced growth and transactivation in MCF-7 Promoter analysis studies in breast cancer cells have identified and ZR-75 human breast cancer cells. Transfection of Sp1 GC-rich sites required for hormone activation of several genes iRNA in MCF-7 or ZR-75 cells for 36 – 44 h decreased Sp1 including E2F1, DNA polymerase , cyclin D1, insulin-like protein (50 –70%) in nuclear extracts, and immunohisto- growth factor-binding protein 4, retinoic acid receptor 1, ca- chemical analysis showed that the Sp1 protein in trans- thepsin D, vascular endothelial growth factor, c-fos, heat shock fected MCF-7 cells was barely detectable. In cell cycle protein 27, bcl-2, thymidylate synthase, and adenosine deami- progression studies in MCF-7 cells, decreased Sp1 protein nase (20 –27, 29 –31). Studies in other cell lines have also dem- was accompanied by a decrease in cells in the S phase and onstrated a role for ER/Sp1 activation of the progesterone an increase in cells in G /G , and estrogen-induced G /G 0 1 0 1 receptor, epidermal growth factor receptor, telomerase, and 3 S phase progression was inhibited in cells treated with receptor for advanced glycation end products (32–35). Activa- iRNA for Sp1. Sp1 iRNA also specifically blocked basal tion of ER/Sp1 does not require the DNA binding domain of and estrogen-induced transactivation in cells transfected ER (promoter DNA-independent) and is primarily dependent with a GC-rich construct linked to a luciferase reporter on the activation function-1 (AF1) of ER (30), whereas DNA- gene (pSp1 ), and this was accompanied by decreased Sp1 dependent activation through ER binding to estrogen response binding to this GC-rich promoter as determined in gel mobility shift and chromatin immunoprecipitation as- elements (EREs) is primarily dependent on AF2 of ER. says. These results clearly demonstrate the key role of the Recent studies have demonstrated that RNA interference Sp1 protein in basal and estrogen-induced growth and through small inhibitory RNAs (iRNAs) targeted to endoge- gene expression in breast cancer cells. nous or heterologous genes can be used to suppress intracellu- lar expression of these genes in mammalian cells, and this technique is well suited for mechanistic studies on gene/protein Sp1 is a member of the Sp and Kru ¨ ppel-like family of tran- function (36 – 42). This study investigates the role of Sp1 pro- scription factors that bind GC and CACCC boxes to regulate tein in mediating hormone-responsiveness in MCF-7 cells us- gene expression (1–3). Sp1 is widely expressed in multiple ing sequence-specific duplexes of 21 nucleotides targeted to Sp1 tissues (4), and targeted disruption of Sp1 in mice results in mRNA as well as Lamin B1 and the heterologous firefly lucif- retarded development and embryo-lethality (5). Sp1 interacts erase gene (GL2) mRNAs. Transfection of iRNA for Sp1 (iSp1) with GC-rich Sp1 binding sites in multiple promoters to regu- decreases (40 – 60%) the expression of nuclear Sp1 protein in late gene expression, and there are an increasing number of ER-positive MCF-7 and ZR-75 human breast cancer cell ex- studies showing that Sp1 interacts with other nuclear proteins, tracts. In transfected cells, Sp1 protein is barely detectable by including promoter-bound transcription factors, to attenuate immunofluorescence, and both basal and estrogen-inducible tissue-specific expression of selected genes (1–3). For example, transactivation is decreased in cells transfected with iSp1 and Sp1 and NFY cooperatively interact to regulate multiple genes a GC-rich construct. These data, combined with results show- through NFY-GC-rich motifs, and both proteins also physically ing that iSp1 inhibits hormone-induced MCF-7 cell cycle pro- interact (6 –10). Sp1 also binds estrogen receptor  (ER) and gression from G /G to S phase, demonstrate that ER/Sp1- 0 1 mediated transactivation plays a major role in ER-positive breast cancer cell growth. * This study was supported in part by National Institutes of Health Grants CA96676 and ES09106, the Texas Agricultural Experiment Station, and the Sid Kyle endowment. The costs of publication of this MATERIALS AND METHODS article were defrayed in part by the payment of page charges. This Cell Lines—MCF-7 and ZR-75 cells were obtained from the American article must therefore be hereby marked “advertisement” in accordance Type Culture Collection (ATCC, Manassas, VA). DME/F12 with and with 18 U.S.C. Section 1734 solely to indicate this fact. without phenol red, 100 antibiotic/antimycotic solution, propidium To whom correspondence should be addressed: Dept. of Veterinary idodide, and E2 were purchased from Sigma. Fetal bovine serum was Physiology and Pharmacology, Texas A&M University, 4466 TAMU, purchased from Intergen (Purchase, NY). [- P]ATP (300Ci/mmol) was Vet. Res. Bldg. 409, College Station, TX 77843-4466. Tel.: 979-845-5988; obtained from PerkinElmer Life Sciences. Poly(dI-dC) and T4 polynu- Fax: 979-862-4929; E-mail: ssafe@cvm.tamu.edu. cleotide kinase were purchased from Roche Molecular Biochemicals The abbreviations used are: ER, estrogen receptor ; ERE, estro- (Indianapolis, IN). Antibodies for proteins Sp1, Lamin B1, and ER gen response elements; i, inhibitory; FACS, fluorescence-activated cell proteins were obtained from Santa Cruz Biotechnology (Santa Cruz, sorter; PI, propidium idodide; EMSA, electrophoretic mobility shift CA). Human ER expression plasmid was provided by Dr. Ming-Jer assay; PBS, phosphate-buffered saline, ChIP, chromatin immunopre- cipitation assay; LMN, Lamin B1. Tsai, Baylor College of Medicine (Houston, TX). The pSp1 construct This paper is available on line at http://www.jbc.org 28815 This is an Open Access article under the CC BY license. 28816 Inhibitory RNA for Sp1 Inhibits MCF-7 Cell Proliferation FIG.1. Interfering RNA for Sp1 (iSp1) decreases Sp1 protein in MCF-7 and ZR-75 cells. A, effects on Sp1 protein in MCF-7 cells. Cells were transfected with iSp1 and iLMN, and nuclear extracts were analyzed for Sp1 and ER proteins by Western blot analysis as described under “Materials and Methods.” Results are means  S.D. for three replicate determinations for each treatment group, and a significant (p  0.05) decrease in Sp1 protein levels was observed. B, effects on Lamin B1 in MCF-7 cells. Cells were treated as described in A, and Lamin B1 and ER proteins were detected by Western blot analysis. Treatment with iLMN significantly (p  0.05) decreased Lamin B1 protein. C, ZR-75 cells. Experiments were carried out as described in MCF-7 cells (A), and iSp1 significantly (p  0.05) decreased Sp1 protein in ZR-75 cells. contains three consensus Sp1 binding sites and the pERE construct medium supplemented with 5% fetal bovine serum. After 16 –20 h when contains three EREs. The oligonucleotides were linked to the bacterial cells were 50 – 60% confluent, iRNA duplexes and/or reporter gene con- luciferase gene and cloned into BamHI-HindIII-cut XP-2 plasmid ob- structs were transfected using LipofectAMINE Plus Reagent (Invitro- tained from ATCC. Lysis buffer, luciferase reagent, and RNase were gen). The effects of iSp1 on hormone-induced transactivation was in- obtained from Promega Corp. (Madision, WI). All other chemicals and vestigated in MCF-7 cells treated with 10 nM E2 and cotransfected with biochemicals were of the highest quality available from commercial pSp1 (500 ng) or pERE (500 ng) and ER expression plasmid (200 ng). 3 3 sources. iRNAs were prepared by IDT (Coralville, IA) and targeted the Based on the results of preliminary studies, 0.75 g of iRNA duplex was coding region 153–173, 672– 694, and 1811–1833 relative to the start transfected in each well to give a final concentration of 50 nM. Cells codon of GL2, Lamin B1 (LMN), and Sp1 genes, respectively. Single- were harvested 36 – 44 h after transfection by manual scraping in 1 stranded RNAs were annealed by incubating 20 M of each strand in lysis buffer (Promega). For whole cell extracts, cells were frozen in annealing buffer (100 mM potassium acetate, 30 mM HEPES buffer at liquid nitrogen for 30 s, vortexed for 30 s, and centrifuged at 12,000 pH 7.4, 2 mM magnesium acetate) for 1 min at 90 °C followed by1hat g for 1 min. Lysates were assayed for luciferase activity using luciferase 37 °C. The iRNA duplexes used in this study are indicated as follows: assay reagent (Promega). -Galactosidase activity was measured using Tropix Galacto-Light Plus assay system (Tropix, Bedford, MA) in a GL2 5-CGUACGCGGAAUACUUCGATT-3 Lumicount microwell plate reader (Packard Instrument Co.). For nu- 3-TTGCAUGCGCCUUAUGAAGCU-5 clear extracts, cells were washed in PBS (2), scraped in 1 ml of 1 LMN1 5-AACGCGCUUGGUAGAGGUGGATT-3 lysis buffer, incubated at 4 °C for 15 min, and centrifuged at 14,000 3-TTUUGCGCGAACCAUCUCCACCU-5 g for 1 min at 20 °C. Cell pellets were initially washed in 1 ml of lysis buffer (3). Lysis buffer supplemented with 500 mM KCl was then Sp1 5-AUCACUCCAUGGAUGAAAUGATT-3 added to the cell pellet and incubated for 45 min at 4 °C with frequent 3-TTUAGUGAGGUACCUACUUUACU-5 vortexing. Nuclei were pelleted by centrifugation at 14,000  g for 1 Transfection of MCF-7 and ZR-75 Cells and Preparation of Nuclear min at 4 °C, and aliquots of supernatant were stored at 80 °C and Extracts—Cells were cultured in 6-well plates in 2 ml of DME/F12 used for Western blot analysis and gel shift assays. Inhibitory RNA for Sp1 Inhibits MCF-7 Cell Proliferation 28817 FIG.2. Binding of [ P]Sp1 with nuclear extracts from breast cancer cells treated with iSp1 or iLMN. MCF-7 (A) or ZR-75 (B) cells were treated with solvent, iSp1, or iLMN, and binding of nuclear extracts to [ P]Sp1 was determined in gel mobility shift assays as described under “Materials and Methods.” C, chromatin immunoprecipitation assay. MCF-7 cells were transfected with pSp1 and iSp1 or iGL2, and analysis of Sp1 and Sp3 immunoprecipitable complexes associated with the transfected GC-rich construct were determined by chromatin immunoprecipitation assay/PCR as described under “Materials and Methods.” Western Immunoblot—An aliquot of nuclear protein (30 g) was autoradiography film. Band intensities were determined by a scanning diluted with loading buffer, boiled, and loaded on a 7.5% SDS-polyacryl- laser densitometer (Sharp Electronics Corp., Mahwah, NJ) using amide gel. Samples were electrophoresed at 150 –180 V for 3–4h,and Zero-D Scanalytics software (Scanalytics Corp., Billerica, MA). separated proteins were transferred to polyvinylidene difluoride mem- FACS Analysis—Cells were transfected with iRNAs for Sp1 or GL2 brane (Bio-Rad, Hercules, CA) in buffer containing 48 mM Tris-HCl, 29 and, after 20 –24 h, treated with Me SO or 20 nM E2 for 18 –20hin mM glycine, and 0.025% SDS. Proteins were detected by incubation with serum-free medium. Cells were then trypsinized and 2  10 cells polyclonal primary antibodies Sp1-PEP2, Lamin B1-C20, and ER-G20 were centrifuged and resuspended after removal of trypsin in 1 ml of (all 1:1000 dilution) against Sp1, Lamin B1, and ER proteins, respec- staining solution containing 50 g/ml propidium iodide, 4 mM sodium tively, followed by blotting with horseradish peroxidase-conjugated an- citrate, 30 units/ml RNase, and 0.1% Triton X-100, pH 7.8. Cells were ti-rabbit (for Sp1 and ER) or anti-goat (for Lamin B) secondary anti- incubated at 37 °C for 10 min, and then prior to FACS analysis sodium body (1:5000 dilution). Blots were then exposed to chemiluminescent chloride was added to give a final concentration of 0.15 M. Cells were substrate (PerkinElmer Life Sciences) and placed in Kodak X-Omat AR analyzed on a FACSCalibur flow cytometer (BD PharMingen) using 28818 Inhibitory RNA for Sp1 Inhibits MCF-7 Cell Proliferation FIG.3. Immunoflourescence of Sp1 and Lamin B in MCF-7 cells trans- fected with iSp1 and iLMN. MCF-7 cells were untreated (A and E), trans- fected with iSp1 (H), iLMN (D and G), and stained with Sp1 (F–H) or Lamin B (B–D) antibodies. Immunofluorescence was de- termined as described under “Materials and Methods.” CellQuest (BD PharMingen) acquisition software. Propidium iodide (85 mM potassium chloride, 0.5% Nonidet P-40, 1 nM phenylmethylsul- fluorescence was collected through a 585/42-nm bandpass filter, and list fonyl fluoride, 5 g/ml leupeptin, and aprotinin at pH 8.0), homoge- mode data were acquired on a minimum of 12,000 single cells defined by nized, and nuclei were isolated by centrifugation at 1500  g for 30 s. a dot plot of PI-width versus PI-area. Data analysis was performed in Nuclei were then resuspended in sonication buffer (1% SDS, 10 nM ModFit LT (Verity Software House, Topsham, ME) using PI-width EDTA, 50 mM Tris at pH 8.1) and sonicated for 45– 60 s to obtain versus PI-area to exclude cell aggregates. FlowJo (Treestar, Inc., Palo chromatin with appropriate fragment lengths (500 –1000 bp). The son- Alto, CA) was used to generate plots shown in the figures. icated extract was then centrifuged at 15,000  g for 10 min at 0 °C, Electrophoretic Mobility Shift Assay (EMSA)—Consensus Sp1 oligo- aliquoted, and stored at 70 °C until used. The cross-linked chromatin nucleotide (28, 30) was synthesized and annealed, and 5-pmol aliquots preparations were diluted in buffer (1% Triton X, 100 mM sodium were 5-end-labeled using T4 kinase and [- P]ATP. A 30-l EMSA chloride, 0.5% SDS, 5 mM EDTA and Tris at pH 8.1), and 20 lof reaction mixture contained 100 mM KCl, 3 g of crude nuclear protein, ultralink protein A or G or A/G beads were added per 100 l of chro- 1 g poly(dI-dC), with or without unlabeled competitor oligonucleotide, matin, and incubated for4hat4 °C. Beads were collected by centrifu- and 10 fmol of radiolabeled probe. After incubation for 20 min on ice, gation, and salmon sperm DNA, specific antibodies, and 20 l of ultra- antibodies against Sp1 protein were added and incubated another 20 link beads were added to the supernatant. The mixture was incubated min on ice. Protein-DNA complexes were resolved by 5% polyacryl- for6hat4 °C. An aliquot was treated at 65 °C to reverse the cross- amide gel electrophoresis in 1 Tris borate/EDTA buffer (0.09 M Tris- links, extracted with phenol/chloroform, and DNA was precipitated base, 0.09 M boric acid, 2 mM EDTA, pH 8.3) at 120 V at 4 °C for 2–3h. with ethanol. This aliquot was used as an input control. Immunopre- Specific DNA-protein and antibody-supershifted complexes were ob- cipitated samples were then centrifuged. Beads were resuspended in served as retarded bands in the gel. dialysis buffer, vortexed for 5 min at 20 °C, and centrifuged at 15,000 Immunocytochemistry—MCF-7 cells were seeded in Lab-Tek Cham- g for 10 s. Beads were then resuspended in immunoprecipitation buffer ber slides (Nalge Nunc International, Naperville, IL) at 100,000 cells/ (11 mM Tris, 500 mM lithium chloride, 1% Nonidet P-40, 1% deoxycholic well in DME/F12 medium supplemented with 5% fetal bovine serum, acid at pH 8,0) and vortexed for 5 min at 20 °C. Procedures with the and after 14 h cells were transferred into serum-free medium for 8 –10 dialysis and immunoprecipitation buffers were repeated (3– 4 times), h. Cells were then transfected with iRNAs, and after 36 – 44 h the media and beads were then resuspended in elution buffer (50 nM NaHCO ,1% chamber was detached and the remaining glass slides were washed in SDS, 1.5 g/ml sonicated salmon sperm DNA), vortexed, and incubated Dulbecco’s PBS. After washing, the glass slides were fixed with cold at 65 °C for 15 min. Supernatants were then isolated by centrifugation (20 °C) methanol for 10 min, and then slides were washed in 0.3% and incubated at 65 °Cfor6hto reverse protein-DNA cross-links. PBS/Tween for 5 min (2) before blocking with 5% rabbit or goat serum Wizard PCR kits (Promega) were used for additional DNA cleanup. A in antibody dilution buffer (stock solution: 100 ml of PBS/Tween,1gof portion of the purified immunoprecipitated DNA and 0.2% of the input bovine serum albumin, 45 ml of glycerol, pH 8.0) for1hat20 °C. After control were used for [-P ]dCTP incorporation PCR. One-fourth of a removal of the blocking solution, rabbit Sp1-PEP2 or goat Lamin B1 microliter of [-P ]dCTP (3000 Ci/mmol) was added to a 25-l PCR polyclonal antibodies were added in antibody dilution buffer (1:200) and reaction (3% Me SO), 1 M betaine, and 1.5 mM magnesium chloride) and incubated for 12 h at 4 °C. Slides were washed for 10 min with 0.3% subjected to one cycle of 95 °C  5 min, 5 cycles of 95 °C  30 s, 60 °C Tween in 0.02 M PBS (3) and incubated with fluorescein isothiocya- 30 s, 5 cycles of 95 °C  30 s, 55 °C  30 s, 72 °C  30 s, and 5 cycles nate-conjugated anti-rabbit or anti-goat secondary antibodies (1:1000 of 95 °C  30 s, 48 °C  30 s, 72 °C  30 s, followed by one cycle at 72 °C dilution) for2hat20 °C. Slides were then washed for 10 min in 0.3% for 4 min. Reactions were loaded on a 10 –15% non-denaturing acryl- PBS-Tween (4). Slides were mounted in ProLonged antifading me- amide gel. The gel was then dried and exposed to a phosphor screen for dium (Molecular Probes, Inc., Eugene, OR), and cover slips were sealed 24 h. The primers used for PCR of the GC-rich region of pSp1 are using Nailslicks nail polish (Noxell Corp., Hunt Valley, MD). Fluores- indicated as follows: pxp2-luc-Fw (6128), 5-GTTTGTCCAAACTCAT- cence imaging was performed using Carlzeiss Axiophoto 2 (Calzeiss, CAATG-3; Rv (105), 5-CTTTATGTTTTTGGCGTCTTC-3. Inc., Thornwood, NY). Images were captured using Adobe Photoshop Statistical Analysis—Statistical significance was determined by 5.5 using identical camera settings. analysis of variance and Scheffe’s test, and the levels of probability are Chromatin Immunoprecipitation Assay (ChIP)—Cells were trans- noted. The results are expressed as means  S.D. for at least three fected with iSp1 or iGL2 for 36 h, then treated with Me SO. MCF-7 cells separate (replicate) experiments for each treatment. were then collected, suspended in 1 PBS with 1 mM phenylmethylsul- fonyl fluoride, and formaldehyde was added to the medium to give a 1% RESULTS solution that was incubated with shaking for 10 min at 20 °C. Glycine iSp1 Specifically Decreases Nuclear Sp1 Protein Levels in was then added (0.125 M) and, after further incubation for 10 min, cells ER-positive Human Breast Cancer Cells—Results of prelimi- were collected by centrifugation and washed with PBS and 1 nM phen- ylmethylsulfonyl fluoride. Cells were then resuspended in swell buffer nary studies indicate that iSp1 and iLMN were most effective Inhibitory RNA for Sp1 Inhibits MCF-7 Cell Proliferation 28819 FIG. 4. Effects of iLMN, iSp1, and iGL2 on luciferase activity in MCF-7 cells transfected with pSp1 and treated with Me SO or E2. A, effects of inhibitor RNAs on basal activity. MCF-7 cells were transfected with pSp1 alone or in combination with iLMN, iGL2, or iSp1 and treated with Me SO. Luciferase ac- tivity was determined as described under “Materials and Methods.” B, iSp1-medi- ated inhibition of transactivation in cells transfected with pSp1 . Cells were trans- fected with pSp1 and iSp1, treated with Me SO or 10 nM E2, and luciferase activ- ity was determined as described under “Materials and Methods.” C, effects of iSp1 on cells transfected with pERE . Cells were transfected with pERE and iLMN, iGL2, or iSp1 treated with Me SO or 10 nM E2, and luciferase activity was determined as described under “Materials and Methods.” Results summarized in A, B, and C are means  S.D. for three rep- licate determinations for each treatment group, and significant (p  0.05) de- creases in activity are indicated by an asterisk. 28820 Inhibitory RNA for Sp1 Inhibits MCF-7 Cell Proliferation FIG.5. Effects of iSp1 on hormone-induced cell cycle progression in MCF-7 cells. Serum-starved MCF-7 cells were treated with Me SO or E2 alone or cotransfected with iGL2 and iSp1, and the percent of distribution of cells in G /G , S, and G /M were determined by FACS analysis 1 0 2 as described under “Materials and Methods.” Similar results were observed in a duplicate analysis. at decreasing cellular protein levels by treating cells for 36 – 44 ciency in MCF-7 cells, suggesting that iSp1 is highly effective h with 0.75 g of the duplex oligonucleotides. The results in decreasing Sp1 expression in transfected cells. This was illustrated in Fig. 1, A and B show that transfection of iSp1 in further investigated in MCF-7 cells by immunofluorescence MCF-7 cells significantly decreased Sp1 protein by 60% in analysis of Sp1 or lamin protein in MCF-7 transfected with nuclear extracts, whereas immunoreactive Lamin B1 and ER iSp1 or iLMN (Fig. 3). Panels A and E are control panels where levels were unchanged. In contrast, transfection of iLMN de- the primary antibody for lamin (panel A) or Sp1 (panel E) has creased Lamin B1 but not Sp1 or ER protein levels, thus been omitted. Panel C is a control for lamin (iLMN) showing demonstrating the specificity of the iRNAs. The results sum- immunofluorescence of Lamin B and phase contrast (panel B). marized in Fig. 1C confirm that iSp1 (but not iLMN) also In cells transfected with iLMN, most of the cells exhibited significantly decreased Sp1 protein in ER-positive ZR-75 cells. either significantly decreased Lamin B expression (transfected The effects of iRNAs on nuclear protein levels were also inves- cells) or lamin expression was unchanged (non-transfected tigated in gel mobility shift assays using extracts from MCF-7 cells). Sp1 staining was observed in untreated MCF-7 cells or ZR-75 cells (Fig. 2, A and B) and a consensus GC-rich (panel F) or in cells transfected with iLMN (panel G); however, oligonucleotide [ P]Sp1 that binds Sp1 and other Sp1 family in cells transfected with iSp1, there was a marked decrease of proteins. Incubation of nuclear extracts from MCF-7 cells with Sp1 staining in most cells, whereas the non-transfected cells [ P]Sp1 gave a profile of retarded bands (lane 2) associated were essentially unchanged. These data demonstrate that with Sp1- and Sp3-DNA complexes (28). The intensity of the transfected iSp1 but not iLMN were highly effective in decreas- former complex was decreased after incubation with unlabeled ing cellular expression of Sp1, and this accounts for the de- Sp1 oligonucleotide (lane 5) and supershifted with Sp1 antibod- creases in Sp1 protein in nuclear extracts (Figs. 1 and 2). ies (lane 6). In cells transfected with iSp1, there was a decrease The results in Fig. 4A summarize the effects of iLMN, iGL2, in retarded band intensity (lane 4), whereas iLMN did not and iSp1 on luciferase activity in MCF-7 cells transfected with affect retarded band intensities. The results obtained for ZR-75 pSp1 and the iRNAs. iLMN did not significantly decrease cells (Fig. 2B) were similar to those observed in MCF-7 cells activity, whereas iGL2 (which is targeted to the luciferase and confirm the effectiveness and specificity of iSp1 for selec- mRNA) and iSp1 both inhibited luciferase activity. In this tively decreasing Sp1 protein in breast cancer cells. study (Fig. 4, A and B), there was a 60 –77% decrease in basal We have also used a chromatin immunoprecipitation assay activity in cells transfected with iSp1. Moreover, E2 induced to further investigate the in situ effects of iSp1 on Sp1-DNA luciferase activity in MCF-7 cells transfected with pSp1 as interactions. MCF-7 cells were cotransfected with iSp1 or iGL2 previously described (28), and in cells cotransfected with iSp1 and a construct containing three tandem GC-rich Sp1 binding there was a 80% decrease in hormone-induced transactiva- sites (pSp1 ), and after 36 – 44 h, cells were treated with form- tion. Thus, iSp1 inhibited both basal and E2 induced luciferase aldehyde to cross-link DNA-bound proteins. After immunopre- activity in MCF-7 cells transfected with pSp1 . In contrast, cipitation with Sp1 or Sp3 antibodies and removal of the cross- hormone-induced transactivation in MCF-7 cells transfected links, PCR was used to identify the GC-rich region of pSp1 as with pERE was not affected by cotransfection with iLMN or 3 3 part of the immunoprecipitable complexes. The results showed iSp1, whereas iGL2 decreased activity in cells treated with that iSp1 decreased interaction of Sp1 with the GC-rich pro- Me SO or E2 (Fig. 4C). Thus, iSp1 specifically blocks hormone- moter compared with that observed in cells transfected with induced transactivation in cells transfected with pSp1 but not iGL2, whereas the intensity of PCR products was similar for pERE . Sp3 immunoprecipitable complexes. Thus, results of Western iSp1 Inhibits Hormone-induced MCF-7 Cell Cycle Progres- blots, gel mobility shift, and ChIP demonstrate a significant sion—Promoter regions in several genes associated with cell (40 – 60%) decrease in Sp1 protein in breast cancer cells trans- proliferation contain E2-responsive GC-rich motifs (20 –31); fected with iSp1. however, the role of ER/Sp1 in mediating cell growth can only Sp1 Protein Expression, Sp1, and ER/Sp1-mediated Trans- be inferred from these studies. The role of Sp1 in hormone- activation in MCF-7 Cells Transfected with iSp1—Transfection induced cell cycle progression was further investigated to de- with LipofectAMINE results in 40 – 60% transfection effi- termine the effects of iSp1 and iGL2 (a control) on distribution Inhibitory RNA for Sp1 Inhibits MCF-7 Cell Proliferation 28821 of MCF-7 cells in G /G ,G -M, and S phases after treatment ingly, immunofluorescence studies indicate that Sp1 protein is 0 1 2 barely detectable in transfected cells (Fig. 3). The high effi- with solvent (Me SO) or 20 nM E2 for 18 –20 h. At this time point, iRNA for Sp1 increased the percent of solvent-treated ciency of iSp1 for ablating Sp1 protein in transfected cells was cells in G observed in MCF-7 cells cotransfected with iSp1 and pSp1 /G from 75.3 to 78.3% and decreased the percent in ,an 0 1 3 S phase (from 15.1 to 12.1). In a parallel study in untreated E2-responsive GC-rich construct that serves as a surrogate for cells at an earlier time point (8 –10 h), a 5% decrease in cells in other GC-rich E2-responsive gene promoters (Fig. 4). In these S phase and a similar increase in cells in G /G was observed transfection studies, iSp1 significantly decreased both basal 0 1 and E2-induced luciferase activities confirming the role of ER/ (data not shown). More dramatic changes were observed for the effects of iSp1 on E2-induced proliferation of MCF-7 cells. For Sp1 in ligand-activated transcription. example, in cells treated with Me Treatment of growth-arrested MCF-7 cells with E2 results in SO or 20 nM E2, the percent of cells in G /G :S phase was 75.3:9.57% or 66.1:23.7%, respec- cell cycle progression that is characterized by a decrease in cells 0 1 in G tively, showing a dramatic increase in G /G 3 S progression /G and an increase in cells in S phase (43, 44) (Fig. 5). In 0 1 0 1 after treatment with E2, and this has been observed in other untreated cells, iSp1 further increased the percent of cells in studies (43, 44). In contrast, the percent of cells in G G /G :S /G (from 75.3 to 78.3%) and decreased the number of cell in 0 1 0 1 phase in cells treated with iSp1 was 71.9:17.3%, indicating that S phase (from 15.1 to 12.1%). Because FACS analysis was hormone-induced cell cycle progression was markedly de- carried out on the total cell population (transfected and non- creased by ablating cellular expression of Sp1 protein, whereas transfected), the response of MCF-7 cells to transfected iSp1 transfection of the control iGL2 did not affect cell cycle progres- demonstrates the important role of Sp1-regulated genes in sion. These results demonstrate for the first time that Sp1 basal growth of these cells. The effects of iSp1 were more protein and ER/Sp1-mediated transactivation are important dramatic in reversing hormone-induced cell cycle progression for hormone-induced proliferation of MCF-7 cells. and blocking a high proportion of these cells from progression to S phase. These data are consistent with the results of pre- DISCUSSION vious studies showing that cyclin D1 and other genes important The development of genetic technologies to regulate or delete for cell proliferation are regulated by ER/Sp1 (21, 25, 26, 28, expression of endogenous genes has been extensively used to 29). Future studies will use iRNAs to further investigate the probe the role of specific genes on biological function. For ex- role of Sp1, other Sp-like proteins, and coregulatory factors on ample, the generation of knock-out/knock-in mice and the over- the growth of MCF-7 and other hormone-dependent cell lines expression of genes in transgenic animal models has provided and to identify key genes that are integral for these responses. unique insights on gene function in normal physiology and REFERENCES disease processes. RNA interference by double-stranded RNA 1. Lania, L., Majello, B., and De Luca, P. (1997) Int. J. Biochem. Cell Biol. 29, involves the sequence-specific post-transcriptional silencing of 1313–1323 genes that has been widely described and used in plants and 2. Philipsen, S., and Suske, G. (1999) Nucleic Acids Res. 27, 2991–3000 3. Black, A. R., Black, J. D., and Azizkhan-Clifford, J. (2001) J. Cell. Physiol. 188, animals (37, 38, 40, 41). It has recently been shown that small 143–160 interfering RNA duplexes (21–25 nucleotides) targeted to spe- 4. Saffer, J. D., Jackson, S. P., and Annarella, M. B. (1991) Mol. Cell. Biol. 11, 2189 –2199 cific genes can now be introduced into mammalian cells in 5. Marin, M., Karis, A., Visser, P., Grosveld, F., and Phillipsen, S. (1997) Cell 89, culture to decrease RNA/protein expression (36 – 42). Elbashir 619 – 628 et al. (36) recently reported iRNA duplexes for endogenous and 6. Roder, K., Wolf, S. S., Larkin, K. J., and Schweizer, M. (1999) Gene (Amst.) 234, 61– 69 exogenous genes decreased their corresponding protein and/or 7. Liang, F., Schaufele, F., and Gardner, D. G. (2001) J. Biol. Chem. 276, protein-dependent activities in several mammalian cell lines 1516 –1522 8. Inoue, T., Kamiyama, J., and Sakai, T. (1999) J. Biol. Chem. 274, 32309 –32317 including NIH 3T3, HeLa, COS-7, and 293 cells. 9. Xiong, S., Chirala, S. S., and Wakil, S. J. (2000) Proc. Natl. Acad. Sci. U. S. A. This study has used the iRNA approach for investigating the 97, 3948 –3953 role of Sp1 protein in the growth and hormone-responsiveness 10. Hu, Z., Jin, S., and Scotto, K. W. (2000) J. Biol. Chem. 275, 2979 –2985 11. Porter, W., Saville, B., Hoivik, D., and Safe, S. (1997) Mol. Endocrinol. 11, of MCF-7 human breast cancer cells. Although Sp1 is impor- 1569 –1580 tant for basal transcription of genes involved in cell growth, 12. Pipao ´ n, C., Tsai, S. Y., and Tsai, M. J. (1999) Mol. Cell. Biol. 19, 2734 –2745 13. Suzuki, Y., Shimada, J., Shudo, K., Matsumura, M., Crippa, M. P., and expression of several cell cycle-regulated genes such as dihy- Kojima, S. (1999) Blood 93, 4264 – 4276 drofolate reductase and hypoxanthine/guanine phosphoribosyl 14. Owen, G. I., Richer, J. K., Tung, L., Takimoto, G., and Horwitz, K. B. (1998) transferase were unaffected in gestation day 8.5-day-old em- J. Biol. Chem. 273, 10696 –10701 15. Lu, S., Jenster, G., and Epner, D. E. (2000) Mol. Endocrinol. 14, 753–760 bryos (5). In contrast, transfection of GC-rich Sp1 oligonucleo- 16. Liu, Z., and Simpson, E. R. (1999) Mol. Cell. Endocrinol. 153, 183–196 tide decoys into A549 human lung adenocarcinoma and U251 17. Sugawara, T., Saito, M., and Fujimoto, S. (2000) Endocrinology 141, 2895–2903 human glioblastoma cells blocked expression of several genes 18. Curtin, D., Jenkins, S., Farmer, N., Anderson, A. C., Haisenleder, D. J., with GC-rich promoters and suppressed cell growth. This ap- Rissman, E., Wilson, E. M., and Shupnik, M. A. (2001) Mol. Endocrinol. 15, proach and others that target GC-rich sequences suggest that 1906 –1917 19. Shimada, J., Suzuki, Y., Kim, S. J., Wang, P. C., Matsumura, M., and Kojima, Sp1 protein may play an important role in cell growth (45, 46); S. (2001) Mol. Endocrinol. 15, 1677–1692 however, these techniques lack specificity because multiple Sp 20. Wang, F., Hoivik, D., Pollenz, R., and Safe, S. (1998) Nucleic Acids Res. 26, 3044 –3052 family proteins bind GC-rich motifs that may influence the 21. Wang, W., Dong, L., Saville, B., and Safe, S. (1999) Mol. Endocrinol. 13, function of other DNA-bound transcription factors. Research in 1373–1387 this laboratory has identified E2-responsive GC-rich motifs in 22. Xie, W., Duan, R., and Safe, S. (1999) Endocrinology 140, 219 –227 23. Sun, G., Porter, W., and Safe, S. (1998) Mol. Endocrinol. 12, 882– 890 promoters of several genes involved in cell proliferation, and 24. Qin, C., Singh, P., and Safe, S. (1999) Endocrinology 140, 2501–2508 these include cyclin D1, thymidylate synthase, c-fos, E2F1, 25. Duan, R., Porter, W., and Safe, S. (1998) Endocrinology 139, 1981–1990 26. Dong, L., Wang, W., Wang, F., Stoner, M., Reed, J. C., Harigai, M., Kladde, M., bcl2, and DNA polymerase  (20 –31). Several approaches were Vyhlidal, C., and Safe, S. (1999) J. Biol. Chem. 174, 32099 –32107 previously used to demonstrate the role of ER/Sp1 as a tran- 27. Xie, W., Duan, R., Chen, I., Samudio, I., and Safe, S. (2000) Endocrinology 141, scription factor complex, and this study was designed to further 2439 –2449 28. Castro-Rivera, E., Samudio, I., and Safe, S. (2001) J. Biol. Chem. 276, investigate this non-classical mechanism of estrogen action 30853–30861 and its involvement in hormone-induced transactivation and 29. Samudio, I., Vyhlidal, C., Wang, F., Stoner, M., Chen, I., Kladde, M., Barhoumi, R., Burghardt, R., and Safe, S. (2001) Endocrinology 142, cell proliferation. The results in Figs. 1–3 clearly demonstrate 1000 –1008 that transfected iSp1 was highly effective for decreasing ex- 30. Saville, B., Wormke, M., Wang, F., Nguyen, T., Enmark, E., Kuiper, G., pression of Sp1 protein in nuclear extracts, and, not surpris- Gustafsson, J.-A., and Safe, S. (2000) J. Biol. Chem. 275, 5379 –5387 28822 Inhibitory RNA for Sp1 Inhibits MCF-7 Cell Proliferation 31. Safe, S. (2001) Vitam. Horm. 62, 231–252 40. Tuschl, T. (2001) Chem. Biochem. 2, 239 –245 32. Petz, L. N., and Nardulli, A. M. (2000) Mol. Endocrinol. 14, 972–985 41. Hammond, S. M., Caudy, A. A., and Hannon, G. J. (2001) Nat. Rev. Genet. 2, 33. Salvatori, L., Ravenna, L., Felli, M. P., Cardillo, M. R., Russo, M. A., Frati, L., 110 –119 Gulino, A., and Petrangeli, E. (2000) Endocrinology 141, 2266 –2274 42. Harborth, J., Elbashir, S. M., Bechert, K., Tuschl, T., and Weber, K. (2001) 34. Kyo, S., Takakura, M., Kanaya, T., Zhuo, W., Fujimoto, K., Nishio, Y., Orimo, J. Cell Sci. 114, 4557– 4565 A., and Inoue, M. (1999) Cancer Res. 59, 5917–5921 43. Foster, J. S., and Wimalasena, J. (1996) Mol. Endocrinol. 10, 488 – 498 35. Tanaka, N., Yonekura, H., Yamagishi, S., Fujimori, H., Yamamoto, Y., and 44. Prall, O. W. J., Sarcevic, B., Musgrove, E. A., Watts, C. K. W., and Sutherland, Yamamoto, H. (2000) J. Biol. Chem. 275, 25781–25790 R. L. (1997) J. Biol. Chem. 272, 10882–10894 36. Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, 45. Ishibashi, H., Nakagawa, K., Onimaru, M., Castellanous, E. J., Kaneda, Y., T. (2001) Nature 411, 494 – 498 Nakashima, Y., Shirasuna, K., and Sueishi, K. (2000) Cancer Res. 60, 37. Fire, A. (1999) Trends. Genet. 15, 358 –363 6531– 6536 38. Sharp, P. A. (2001) Genes Dev. 15, 485– 490 46. Martin, B., Vaquero, A., Priebe, W., and Portugal, J. (1999) Nucleic Acids Res. 39. Paddison, P. J., Caudy, A. A., and Hannon, G. J. (2002) Proc. Natl. Acad. Sci. 27, 3402–3409 U. S. A. 99, 1443–1448 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Small Inhibitory RNA Duplexes for Sp1 mRNA Block Basal and Estrogen-induced Gene Expression and Cell Cycle Progression in MCF-7 Breast Cancer Cells

Journal of Biological ChemistryAug 1, 2002

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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 32, Issue of August 9, pp. 28815–28822, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Small Inhibitory RNA Duplexes for Sp1 mRNA Block Basal and Estrogen-induced Gene Expression and Cell Cycle Progression in MCF-7 Breast Cancer Cells* Received for publication, April 19, 2002, and in revised form, May 24, 2002 Published, JBC Papers in Press, June 6, 2002, DOI 10.1074/jbc.M203828200 Maen Abdelrahim‡, Ismael Samudio‡, Roger Smith III§, Robert Burghardt¶, and Stephen Safe‡ From the Departments of ‡Veterinary Physiology and Pharmacology, §Veterinary Pathobiology, and ¶Anatomy and Public Health, Texas A&M University, College Station, Texas 77843-4466 Small interfering RNA duplexes containing 21–22 nucle- other members of the nuclear receptor family of transcription otides that mediate sequence-specific mRNA degradation factors (11–19), and research in our laboratory has focused on and inhibitory RNA (iRNA) for Sp1 mRNA were used in the molecular mechanisms of the ligand-dependent activation this study to investigate the role of Sp1 on basal and of ER/Sp1 in breast and endometrial cancer cell lines (20 –31). hormone-induced growth and transactivation in MCF-7 Promoter analysis studies in breast cancer cells have identified and ZR-75 human breast cancer cells. Transfection of Sp1 GC-rich sites required for hormone activation of several genes iRNA in MCF-7 or ZR-75 cells for 36 – 44 h decreased Sp1 including E2F1, DNA polymerase , cyclin D1, insulin-like protein (50 –70%) in nuclear extracts, and immunohisto- growth factor-binding protein 4, retinoic acid receptor 1, ca- chemical analysis showed that the Sp1 protein in trans- thepsin D, vascular endothelial growth factor, c-fos, heat shock fected MCF-7 cells was barely detectable. In cell cycle protein 27, bcl-2, thymidylate synthase, and adenosine deami- progression studies in MCF-7 cells, decreased Sp1 protein nase (20 –27, 29 –31). Studies in other cell lines have also dem- was accompanied by a decrease in cells in the S phase and onstrated a role for ER/Sp1 activation of the progesterone an increase in cells in G /G , and estrogen-induced G /G 0 1 0 1 receptor, epidermal growth factor receptor, telomerase, and 3 S phase progression was inhibited in cells treated with receptor for advanced glycation end products (32–35). Activa- iRNA for Sp1. Sp1 iRNA also specifically blocked basal tion of ER/Sp1 does not require the DNA binding domain of and estrogen-induced transactivation in cells transfected ER (promoter DNA-independent) and is primarily dependent with a GC-rich construct linked to a luciferase reporter on the activation function-1 (AF1) of ER (30), whereas DNA- gene (pSp1 ), and this was accompanied by decreased Sp1 dependent activation through ER binding to estrogen response binding to this GC-rich promoter as determined in gel mobility shift and chromatin immunoprecipitation as- elements (EREs) is primarily dependent on AF2 of ER. says. These results clearly demonstrate the key role of the Recent studies have demonstrated that RNA interference Sp1 protein in basal and estrogen-induced growth and through small inhibitory RNAs (iRNAs) targeted to endoge- gene expression in breast cancer cells. nous or heterologous genes can be used to suppress intracellu- lar expression of these genes in mammalian cells, and this technique is well suited for mechanistic studies on gene/protein Sp1 is a member of the Sp and Kru ¨ ppel-like family of tran- function (36 – 42). This study investigates the role of Sp1 pro- scription factors that bind GC and CACCC boxes to regulate tein in mediating hormone-responsiveness in MCF-7 cells us- gene expression (1–3). Sp1 is widely expressed in multiple ing sequence-specific duplexes of 21 nucleotides targeted to Sp1 tissues (4), and targeted disruption of Sp1 in mice results in mRNA as well as Lamin B1 and the heterologous firefly lucif- retarded development and embryo-lethality (5). Sp1 interacts erase gene (GL2) mRNAs. Transfection of iRNA for Sp1 (iSp1) with GC-rich Sp1 binding sites in multiple promoters to regu- decreases (40 – 60%) the expression of nuclear Sp1 protein in late gene expression, and there are an increasing number of ER-positive MCF-7 and ZR-75 human breast cancer cell ex- studies showing that Sp1 interacts with other nuclear proteins, tracts. In transfected cells, Sp1 protein is barely detectable by including promoter-bound transcription factors, to attenuate immunofluorescence, and both basal and estrogen-inducible tissue-specific expression of selected genes (1–3). For example, transactivation is decreased in cells transfected with iSp1 and Sp1 and NFY cooperatively interact to regulate multiple genes a GC-rich construct. These data, combined with results show- through NFY-GC-rich motifs, and both proteins also physically ing that iSp1 inhibits hormone-induced MCF-7 cell cycle pro- interact (6 –10). Sp1 also binds estrogen receptor  (ER) and gression from G /G to S phase, demonstrate that ER/Sp1- 0 1 mediated transactivation plays a major role in ER-positive breast cancer cell growth. * This study was supported in part by National Institutes of Health Grants CA96676 and ES09106, the Texas Agricultural Experiment Station, and the Sid Kyle endowment. The costs of publication of this MATERIALS AND METHODS article were defrayed in part by the payment of page charges. This Cell Lines—MCF-7 and ZR-75 cells were obtained from the American article must therefore be hereby marked “advertisement” in accordance Type Culture Collection (ATCC, Manassas, VA). DME/F12 with and with 18 U.S.C. Section 1734 solely to indicate this fact. without phenol red, 100 antibiotic/antimycotic solution, propidium To whom correspondence should be addressed: Dept. of Veterinary idodide, and E2 were purchased from Sigma. Fetal bovine serum was Physiology and Pharmacology, Texas A&M University, 4466 TAMU, purchased from Intergen (Purchase, NY). [- P]ATP (300Ci/mmol) was Vet. Res. Bldg. 409, College Station, TX 77843-4466. Tel.: 979-845-5988; obtained from PerkinElmer Life Sciences. Poly(dI-dC) and T4 polynu- Fax: 979-862-4929; E-mail: ssafe@cvm.tamu.edu. cleotide kinase were purchased from Roche Molecular Biochemicals The abbreviations used are: ER, estrogen receptor ; ERE, estro- (Indianapolis, IN). Antibodies for proteins Sp1, Lamin B1, and ER gen response elements; i, inhibitory; FACS, fluorescence-activated cell proteins were obtained from Santa Cruz Biotechnology (Santa Cruz, sorter; PI, propidium idodide; EMSA, electrophoretic mobility shift CA). Human ER expression plasmid was provided by Dr. Ming-Jer assay; PBS, phosphate-buffered saline, ChIP, chromatin immunopre- cipitation assay; LMN, Lamin B1. Tsai, Baylor College of Medicine (Houston, TX). The pSp1 construct This paper is available on line at http://www.jbc.org 28815 This is an Open Access article under the CC BY license. 28816 Inhibitory RNA for Sp1 Inhibits MCF-7 Cell Proliferation FIG.1. Interfering RNA for Sp1 (iSp1) decreases Sp1 protein in MCF-7 and ZR-75 cells. A, effects on Sp1 protein in MCF-7 cells. Cells were transfected with iSp1 and iLMN, and nuclear extracts were analyzed for Sp1 and ER proteins by Western blot analysis as described under “Materials and Methods.” Results are means  S.D. for three replicate determinations for each treatment group, and a significant (p  0.05) decrease in Sp1 protein levels was observed. B, effects on Lamin B1 in MCF-7 cells. Cells were treated as described in A, and Lamin B1 and ER proteins were detected by Western blot analysis. Treatment with iLMN significantly (p  0.05) decreased Lamin B1 protein. C, ZR-75 cells. Experiments were carried out as described in MCF-7 cells (A), and iSp1 significantly (p  0.05) decreased Sp1 protein in ZR-75 cells. contains three consensus Sp1 binding sites and the pERE construct medium supplemented with 5% fetal bovine serum. After 16 –20 h when contains three EREs. The oligonucleotides were linked to the bacterial cells were 50 – 60% confluent, iRNA duplexes and/or reporter gene con- luciferase gene and cloned into BamHI-HindIII-cut XP-2 plasmid ob- structs were transfected using LipofectAMINE Plus Reagent (Invitro- tained from ATCC. Lysis buffer, luciferase reagent, and RNase were gen). The effects of iSp1 on hormone-induced transactivation was in- obtained from Promega Corp. (Madision, WI). All other chemicals and vestigated in MCF-7 cells treated with 10 nM E2 and cotransfected with biochemicals were of the highest quality available from commercial pSp1 (500 ng) or pERE (500 ng) and ER expression plasmid (200 ng). 3 3 sources. iRNAs were prepared by IDT (Coralville, IA) and targeted the Based on the results of preliminary studies, 0.75 g of iRNA duplex was coding region 153–173, 672– 694, and 1811–1833 relative to the start transfected in each well to give a final concentration of 50 nM. Cells codon of GL2, Lamin B1 (LMN), and Sp1 genes, respectively. Single- were harvested 36 – 44 h after transfection by manual scraping in 1 stranded RNAs were annealed by incubating 20 M of each strand in lysis buffer (Promega). For whole cell extracts, cells were frozen in annealing buffer (100 mM potassium acetate, 30 mM HEPES buffer at liquid nitrogen for 30 s, vortexed for 30 s, and centrifuged at 12,000 pH 7.4, 2 mM magnesium acetate) for 1 min at 90 °C followed by1hat g for 1 min. Lysates were assayed for luciferase activity using luciferase 37 °C. The iRNA duplexes used in this study are indicated as follows: assay reagent (Promega). -Galactosidase activity was measured using Tropix Galacto-Light Plus assay system (Tropix, Bedford, MA) in a GL2 5-CGUACGCGGAAUACUUCGATT-3 Lumicount microwell plate reader (Packard Instrument Co.). For nu- 3-TTGCAUGCGCCUUAUGAAGCU-5 clear extracts, cells were washed in PBS (2), scraped in 1 ml of 1 LMN1 5-AACGCGCUUGGUAGAGGUGGATT-3 lysis buffer, incubated at 4 °C for 15 min, and centrifuged at 14,000 3-TTUUGCGCGAACCAUCUCCACCU-5 g for 1 min at 20 °C. Cell pellets were initially washed in 1 ml of lysis buffer (3). Lysis buffer supplemented with 500 mM KCl was then Sp1 5-AUCACUCCAUGGAUGAAAUGATT-3 added to the cell pellet and incubated for 45 min at 4 °C with frequent 3-TTUAGUGAGGUACCUACUUUACU-5 vortexing. Nuclei were pelleted by centrifugation at 14,000  g for 1 Transfection of MCF-7 and ZR-75 Cells and Preparation of Nuclear min at 4 °C, and aliquots of supernatant were stored at 80 °C and Extracts—Cells were cultured in 6-well plates in 2 ml of DME/F12 used for Western blot analysis and gel shift assays. Inhibitory RNA for Sp1 Inhibits MCF-7 Cell Proliferation 28817 FIG.2. Binding of [ P]Sp1 with nuclear extracts from breast cancer cells treated with iSp1 or iLMN. MCF-7 (A) or ZR-75 (B) cells were treated with solvent, iSp1, or iLMN, and binding of nuclear extracts to [ P]Sp1 was determined in gel mobility shift assays as described under “Materials and Methods.” C, chromatin immunoprecipitation assay. MCF-7 cells were transfected with pSp1 and iSp1 or iGL2, and analysis of Sp1 and Sp3 immunoprecipitable complexes associated with the transfected GC-rich construct were determined by chromatin immunoprecipitation assay/PCR as described under “Materials and Methods.” Western Immunoblot—An aliquot of nuclear protein (30 g) was autoradiography film. Band intensities were determined by a scanning diluted with loading buffer, boiled, and loaded on a 7.5% SDS-polyacryl- laser densitometer (Sharp Electronics Corp., Mahwah, NJ) using amide gel. Samples were electrophoresed at 150 –180 V for 3–4h,and Zero-D Scanalytics software (Scanalytics Corp., Billerica, MA). separated proteins were transferred to polyvinylidene difluoride mem- FACS Analysis—Cells were transfected with iRNAs for Sp1 or GL2 brane (Bio-Rad, Hercules, CA) in buffer containing 48 mM Tris-HCl, 29 and, after 20 –24 h, treated with Me SO or 20 nM E2 for 18 –20hin mM glycine, and 0.025% SDS. Proteins were detected by incubation with serum-free medium. Cells were then trypsinized and 2  10 cells polyclonal primary antibodies Sp1-PEP2, Lamin B1-C20, and ER-G20 were centrifuged and resuspended after removal of trypsin in 1 ml of (all 1:1000 dilution) against Sp1, Lamin B1, and ER proteins, respec- staining solution containing 50 g/ml propidium iodide, 4 mM sodium tively, followed by blotting with horseradish peroxidase-conjugated an- citrate, 30 units/ml RNase, and 0.1% Triton X-100, pH 7.8. Cells were ti-rabbit (for Sp1 and ER) or anti-goat (for Lamin B) secondary anti- incubated at 37 °C for 10 min, and then prior to FACS analysis sodium body (1:5000 dilution). Blots were then exposed to chemiluminescent chloride was added to give a final concentration of 0.15 M. Cells were substrate (PerkinElmer Life Sciences) and placed in Kodak X-Omat AR analyzed on a FACSCalibur flow cytometer (BD PharMingen) using 28818 Inhibitory RNA for Sp1 Inhibits MCF-7 Cell Proliferation FIG.3. Immunoflourescence of Sp1 and Lamin B in MCF-7 cells trans- fected with iSp1 and iLMN. MCF-7 cells were untreated (A and E), trans- fected with iSp1 (H), iLMN (D and G), and stained with Sp1 (F–H) or Lamin B (B–D) antibodies. Immunofluorescence was de- termined as described under “Materials and Methods.” CellQuest (BD PharMingen) acquisition software. Propidium iodide (85 mM potassium chloride, 0.5% Nonidet P-40, 1 nM phenylmethylsul- fluorescence was collected through a 585/42-nm bandpass filter, and list fonyl fluoride, 5 g/ml leupeptin, and aprotinin at pH 8.0), homoge- mode data were acquired on a minimum of 12,000 single cells defined by nized, and nuclei were isolated by centrifugation at 1500  g for 30 s. a dot plot of PI-width versus PI-area. Data analysis was performed in Nuclei were then resuspended in sonication buffer (1% SDS, 10 nM ModFit LT (Verity Software House, Topsham, ME) using PI-width EDTA, 50 mM Tris at pH 8.1) and sonicated for 45– 60 s to obtain versus PI-area to exclude cell aggregates. FlowJo (Treestar, Inc., Palo chromatin with appropriate fragment lengths (500 –1000 bp). The son- Alto, CA) was used to generate plots shown in the figures. icated extract was then centrifuged at 15,000  g for 10 min at 0 °C, Electrophoretic Mobility Shift Assay (EMSA)—Consensus Sp1 oligo- aliquoted, and stored at 70 °C until used. The cross-linked chromatin nucleotide (28, 30) was synthesized and annealed, and 5-pmol aliquots preparations were diluted in buffer (1% Triton X, 100 mM sodium were 5-end-labeled using T4 kinase and [- P]ATP. A 30-l EMSA chloride, 0.5% SDS, 5 mM EDTA and Tris at pH 8.1), and 20 lof reaction mixture contained 100 mM KCl, 3 g of crude nuclear protein, ultralink protein A or G or A/G beads were added per 100 l of chro- 1 g poly(dI-dC), with or without unlabeled competitor oligonucleotide, matin, and incubated for4hat4 °C. Beads were collected by centrifu- and 10 fmol of radiolabeled probe. After incubation for 20 min on ice, gation, and salmon sperm DNA, specific antibodies, and 20 l of ultra- antibodies against Sp1 protein were added and incubated another 20 link beads were added to the supernatant. The mixture was incubated min on ice. Protein-DNA complexes were resolved by 5% polyacryl- for6hat4 °C. An aliquot was treated at 65 °C to reverse the cross- amide gel electrophoresis in 1 Tris borate/EDTA buffer (0.09 M Tris- links, extracted with phenol/chloroform, and DNA was precipitated base, 0.09 M boric acid, 2 mM EDTA, pH 8.3) at 120 V at 4 °C for 2–3h. with ethanol. This aliquot was used as an input control. Immunopre- Specific DNA-protein and antibody-supershifted complexes were ob- cipitated samples were then centrifuged. Beads were resuspended in served as retarded bands in the gel. dialysis buffer, vortexed for 5 min at 20 °C, and centrifuged at 15,000 Immunocytochemistry—MCF-7 cells were seeded in Lab-Tek Cham- g for 10 s. Beads were then resuspended in immunoprecipitation buffer ber slides (Nalge Nunc International, Naperville, IL) at 100,000 cells/ (11 mM Tris, 500 mM lithium chloride, 1% Nonidet P-40, 1% deoxycholic well in DME/F12 medium supplemented with 5% fetal bovine serum, acid at pH 8,0) and vortexed for 5 min at 20 °C. Procedures with the and after 14 h cells were transferred into serum-free medium for 8 –10 dialysis and immunoprecipitation buffers were repeated (3– 4 times), h. Cells were then transfected with iRNAs, and after 36 – 44 h the media and beads were then resuspended in elution buffer (50 nM NaHCO ,1% chamber was detached and the remaining glass slides were washed in SDS, 1.5 g/ml sonicated salmon sperm DNA), vortexed, and incubated Dulbecco’s PBS. After washing, the glass slides were fixed with cold at 65 °C for 15 min. Supernatants were then isolated by centrifugation (20 °C) methanol for 10 min, and then slides were washed in 0.3% and incubated at 65 °Cfor6hto reverse protein-DNA cross-links. PBS/Tween for 5 min (2) before blocking with 5% rabbit or goat serum Wizard PCR kits (Promega) were used for additional DNA cleanup. A in antibody dilution buffer (stock solution: 100 ml of PBS/Tween,1gof portion of the purified immunoprecipitated DNA and 0.2% of the input bovine serum albumin, 45 ml of glycerol, pH 8.0) for1hat20 °C. After control were used for [-P ]dCTP incorporation PCR. One-fourth of a removal of the blocking solution, rabbit Sp1-PEP2 or goat Lamin B1 microliter of [-P ]dCTP (3000 Ci/mmol) was added to a 25-l PCR polyclonal antibodies were added in antibody dilution buffer (1:200) and reaction (3% Me SO), 1 M betaine, and 1.5 mM magnesium chloride) and incubated for 12 h at 4 °C. Slides were washed for 10 min with 0.3% subjected to one cycle of 95 °C  5 min, 5 cycles of 95 °C  30 s, 60 °C Tween in 0.02 M PBS (3) and incubated with fluorescein isothiocya- 30 s, 5 cycles of 95 °C  30 s, 55 °C  30 s, 72 °C  30 s, and 5 cycles nate-conjugated anti-rabbit or anti-goat secondary antibodies (1:1000 of 95 °C  30 s, 48 °C  30 s, 72 °C  30 s, followed by one cycle at 72 °C dilution) for2hat20 °C. Slides were then washed for 10 min in 0.3% for 4 min. Reactions were loaded on a 10 –15% non-denaturing acryl- PBS-Tween (4). Slides were mounted in ProLonged antifading me- amide gel. The gel was then dried and exposed to a phosphor screen for dium (Molecular Probes, Inc., Eugene, OR), and cover slips were sealed 24 h. The primers used for PCR of the GC-rich region of pSp1 are using Nailslicks nail polish (Noxell Corp., Hunt Valley, MD). Fluores- indicated as follows: pxp2-luc-Fw (6128), 5-GTTTGTCCAAACTCAT- cence imaging was performed using Carlzeiss Axiophoto 2 (Calzeiss, CAATG-3; Rv (105), 5-CTTTATGTTTTTGGCGTCTTC-3. Inc., Thornwood, NY). Images were captured using Adobe Photoshop Statistical Analysis—Statistical significance was determined by 5.5 using identical camera settings. analysis of variance and Scheffe’s test, and the levels of probability are Chromatin Immunoprecipitation Assay (ChIP)—Cells were trans- noted. The results are expressed as means  S.D. for at least three fected with iSp1 or iGL2 for 36 h, then treated with Me SO. MCF-7 cells separate (replicate) experiments for each treatment. were then collected, suspended in 1 PBS with 1 mM phenylmethylsul- fonyl fluoride, and formaldehyde was added to the medium to give a 1% RESULTS solution that was incubated with shaking for 10 min at 20 °C. Glycine iSp1 Specifically Decreases Nuclear Sp1 Protein Levels in was then added (0.125 M) and, after further incubation for 10 min, cells ER-positive Human Breast Cancer Cells—Results of prelimi- were collected by centrifugation and washed with PBS and 1 nM phen- ylmethylsulfonyl fluoride. Cells were then resuspended in swell buffer nary studies indicate that iSp1 and iLMN were most effective Inhibitory RNA for Sp1 Inhibits MCF-7 Cell Proliferation 28819 FIG. 4. Effects of iLMN, iSp1, and iGL2 on luciferase activity in MCF-7 cells transfected with pSp1 and treated with Me SO or E2. A, effects of inhibitor RNAs on basal activity. MCF-7 cells were transfected with pSp1 alone or in combination with iLMN, iGL2, or iSp1 and treated with Me SO. Luciferase ac- tivity was determined as described under “Materials and Methods.” B, iSp1-medi- ated inhibition of transactivation in cells transfected with pSp1 . Cells were trans- fected with pSp1 and iSp1, treated with Me SO or 10 nM E2, and luciferase activ- ity was determined as described under “Materials and Methods.” C, effects of iSp1 on cells transfected with pERE . Cells were transfected with pERE and iLMN, iGL2, or iSp1 treated with Me SO or 10 nM E2, and luciferase activity was determined as described under “Materials and Methods.” Results summarized in A, B, and C are means  S.D. for three rep- licate determinations for each treatment group, and significant (p  0.05) de- creases in activity are indicated by an asterisk. 28820 Inhibitory RNA for Sp1 Inhibits MCF-7 Cell Proliferation FIG.5. Effects of iSp1 on hormone-induced cell cycle progression in MCF-7 cells. Serum-starved MCF-7 cells were treated with Me SO or E2 alone or cotransfected with iGL2 and iSp1, and the percent of distribution of cells in G /G , S, and G /M were determined by FACS analysis 1 0 2 as described under “Materials and Methods.” Similar results were observed in a duplicate analysis. at decreasing cellular protein levels by treating cells for 36 – 44 ciency in MCF-7 cells, suggesting that iSp1 is highly effective h with 0.75 g of the duplex oligonucleotides. The results in decreasing Sp1 expression in transfected cells. This was illustrated in Fig. 1, A and B show that transfection of iSp1 in further investigated in MCF-7 cells by immunofluorescence MCF-7 cells significantly decreased Sp1 protein by 60% in analysis of Sp1 or lamin protein in MCF-7 transfected with nuclear extracts, whereas immunoreactive Lamin B1 and ER iSp1 or iLMN (Fig. 3). Panels A and E are control panels where levels were unchanged. In contrast, transfection of iLMN de- the primary antibody for lamin (panel A) or Sp1 (panel E) has creased Lamin B1 but not Sp1 or ER protein levels, thus been omitted. Panel C is a control for lamin (iLMN) showing demonstrating the specificity of the iRNAs. The results sum- immunofluorescence of Lamin B and phase contrast (panel B). marized in Fig. 1C confirm that iSp1 (but not iLMN) also In cells transfected with iLMN, most of the cells exhibited significantly decreased Sp1 protein in ER-positive ZR-75 cells. either significantly decreased Lamin B expression (transfected The effects of iRNAs on nuclear protein levels were also inves- cells) or lamin expression was unchanged (non-transfected tigated in gel mobility shift assays using extracts from MCF-7 cells). Sp1 staining was observed in untreated MCF-7 cells or ZR-75 cells (Fig. 2, A and B) and a consensus GC-rich (panel F) or in cells transfected with iLMN (panel G); however, oligonucleotide [ P]Sp1 that binds Sp1 and other Sp1 family in cells transfected with iSp1, there was a marked decrease of proteins. Incubation of nuclear extracts from MCF-7 cells with Sp1 staining in most cells, whereas the non-transfected cells [ P]Sp1 gave a profile of retarded bands (lane 2) associated were essentially unchanged. These data demonstrate that with Sp1- and Sp3-DNA complexes (28). The intensity of the transfected iSp1 but not iLMN were highly effective in decreas- former complex was decreased after incubation with unlabeled ing cellular expression of Sp1, and this accounts for the de- Sp1 oligonucleotide (lane 5) and supershifted with Sp1 antibod- creases in Sp1 protein in nuclear extracts (Figs. 1 and 2). ies (lane 6). In cells transfected with iSp1, there was a decrease The results in Fig. 4A summarize the effects of iLMN, iGL2, in retarded band intensity (lane 4), whereas iLMN did not and iSp1 on luciferase activity in MCF-7 cells transfected with affect retarded band intensities. The results obtained for ZR-75 pSp1 and the iRNAs. iLMN did not significantly decrease cells (Fig. 2B) were similar to those observed in MCF-7 cells activity, whereas iGL2 (which is targeted to the luciferase and confirm the effectiveness and specificity of iSp1 for selec- mRNA) and iSp1 both inhibited luciferase activity. In this tively decreasing Sp1 protein in breast cancer cells. study (Fig. 4, A and B), there was a 60 –77% decrease in basal We have also used a chromatin immunoprecipitation assay activity in cells transfected with iSp1. Moreover, E2 induced to further investigate the in situ effects of iSp1 on Sp1-DNA luciferase activity in MCF-7 cells transfected with pSp1 as interactions. MCF-7 cells were cotransfected with iSp1 or iGL2 previously described (28), and in cells cotransfected with iSp1 and a construct containing three tandem GC-rich Sp1 binding there was a 80% decrease in hormone-induced transactiva- sites (pSp1 ), and after 36 – 44 h, cells were treated with form- tion. Thus, iSp1 inhibited both basal and E2 induced luciferase aldehyde to cross-link DNA-bound proteins. After immunopre- activity in MCF-7 cells transfected with pSp1 . In contrast, cipitation with Sp1 or Sp3 antibodies and removal of the cross- hormone-induced transactivation in MCF-7 cells transfected links, PCR was used to identify the GC-rich region of pSp1 as with pERE was not affected by cotransfection with iLMN or 3 3 part of the immunoprecipitable complexes. The results showed iSp1, whereas iGL2 decreased activity in cells treated with that iSp1 decreased interaction of Sp1 with the GC-rich pro- Me SO or E2 (Fig. 4C). Thus, iSp1 specifically blocks hormone- moter compared with that observed in cells transfected with induced transactivation in cells transfected with pSp1 but not iGL2, whereas the intensity of PCR products was similar for pERE . Sp3 immunoprecipitable complexes. Thus, results of Western iSp1 Inhibits Hormone-induced MCF-7 Cell Cycle Progres- blots, gel mobility shift, and ChIP demonstrate a significant sion—Promoter regions in several genes associated with cell (40 – 60%) decrease in Sp1 protein in breast cancer cells trans- proliferation contain E2-responsive GC-rich motifs (20 –31); fected with iSp1. however, the role of ER/Sp1 in mediating cell growth can only Sp1 Protein Expression, Sp1, and ER/Sp1-mediated Trans- be inferred from these studies. The role of Sp1 in hormone- activation in MCF-7 Cells Transfected with iSp1—Transfection induced cell cycle progression was further investigated to de- with LipofectAMINE results in 40 – 60% transfection effi- termine the effects of iSp1 and iGL2 (a control) on distribution Inhibitory RNA for Sp1 Inhibits MCF-7 Cell Proliferation 28821 of MCF-7 cells in G /G ,G -M, and S phases after treatment ingly, immunofluorescence studies indicate that Sp1 protein is 0 1 2 barely detectable in transfected cells (Fig. 3). The high effi- with solvent (Me SO) or 20 nM E2 for 18 –20 h. At this time point, iRNA for Sp1 increased the percent of solvent-treated ciency of iSp1 for ablating Sp1 protein in transfected cells was cells in G observed in MCF-7 cells cotransfected with iSp1 and pSp1 /G from 75.3 to 78.3% and decreased the percent in ,an 0 1 3 S phase (from 15.1 to 12.1). In a parallel study in untreated E2-responsive GC-rich construct that serves as a surrogate for cells at an earlier time point (8 –10 h), a 5% decrease in cells in other GC-rich E2-responsive gene promoters (Fig. 4). In these S phase and a similar increase in cells in G /G was observed transfection studies, iSp1 significantly decreased both basal 0 1 and E2-induced luciferase activities confirming the role of ER/ (data not shown). More dramatic changes were observed for the effects of iSp1 on E2-induced proliferation of MCF-7 cells. For Sp1 in ligand-activated transcription. example, in cells treated with Me Treatment of growth-arrested MCF-7 cells with E2 results in SO or 20 nM E2, the percent of cells in G /G :S phase was 75.3:9.57% or 66.1:23.7%, respec- cell cycle progression that is characterized by a decrease in cells 0 1 in G tively, showing a dramatic increase in G /G 3 S progression /G and an increase in cells in S phase (43, 44) (Fig. 5). In 0 1 0 1 after treatment with E2, and this has been observed in other untreated cells, iSp1 further increased the percent of cells in studies (43, 44). In contrast, the percent of cells in G G /G :S /G (from 75.3 to 78.3%) and decreased the number of cell in 0 1 0 1 phase in cells treated with iSp1 was 71.9:17.3%, indicating that S phase (from 15.1 to 12.1%). Because FACS analysis was hormone-induced cell cycle progression was markedly de- carried out on the total cell population (transfected and non- creased by ablating cellular expression of Sp1 protein, whereas transfected), the response of MCF-7 cells to transfected iSp1 transfection of the control iGL2 did not affect cell cycle progres- demonstrates the important role of Sp1-regulated genes in sion. These results demonstrate for the first time that Sp1 basal growth of these cells. The effects of iSp1 were more protein and ER/Sp1-mediated transactivation are important dramatic in reversing hormone-induced cell cycle progression for hormone-induced proliferation of MCF-7 cells. and blocking a high proportion of these cells from progression to S phase. These data are consistent with the results of pre- DISCUSSION vious studies showing that cyclin D1 and other genes important The development of genetic technologies to regulate or delete for cell proliferation are regulated by ER/Sp1 (21, 25, 26, 28, expression of endogenous genes has been extensively used to 29). Future studies will use iRNAs to further investigate the probe the role of specific genes on biological function. For ex- role of Sp1, other Sp-like proteins, and coregulatory factors on ample, the generation of knock-out/knock-in mice and the over- the growth of MCF-7 and other hormone-dependent cell lines expression of genes in transgenic animal models has provided and to identify key genes that are integral for these responses. unique insights on gene function in normal physiology and REFERENCES disease processes. RNA interference by double-stranded RNA 1. Lania, L., Majello, B., and De Luca, P. (1997) Int. J. Biochem. Cell Biol. 29, involves the sequence-specific post-transcriptional silencing of 1313–1323 genes that has been widely described and used in plants and 2. Philipsen, S., and Suske, G. (1999) Nucleic Acids Res. 27, 2991–3000 3. Black, A. R., Black, J. D., and Azizkhan-Clifford, J. (2001) J. Cell. Physiol. 188, animals (37, 38, 40, 41). It has recently been shown that small 143–160 interfering RNA duplexes (21–25 nucleotides) targeted to spe- 4. Saffer, J. D., Jackson, S. P., and Annarella, M. B. (1991) Mol. Cell. Biol. 11, 2189 –2199 cific genes can now be introduced into mammalian cells in 5. Marin, M., Karis, A., Visser, P., Grosveld, F., and Phillipsen, S. (1997) Cell 89, culture to decrease RNA/protein expression (36 – 42). Elbashir 619 – 628 et al. (36) recently reported iRNA duplexes for endogenous and 6. Roder, K., Wolf, S. S., Larkin, K. J., and Schweizer, M. (1999) Gene (Amst.) 234, 61– 69 exogenous genes decreased their corresponding protein and/or 7. Liang, F., Schaufele, F., and Gardner, D. G. (2001) J. Biol. Chem. 276, protein-dependent activities in several mammalian cell lines 1516 –1522 8. Inoue, T., Kamiyama, J., and Sakai, T. (1999) J. Biol. Chem. 274, 32309 –32317 including NIH 3T3, HeLa, COS-7, and 293 cells. 9. Xiong, S., Chirala, S. S., and Wakil, S. J. (2000) Proc. Natl. Acad. Sci. U. S. A. This study has used the iRNA approach for investigating the 97, 3948 –3953 role of Sp1 protein in the growth and hormone-responsiveness 10. Hu, Z., Jin, S., and Scotto, K. W. (2000) J. Biol. Chem. 275, 2979 –2985 11. Porter, W., Saville, B., Hoivik, D., and Safe, S. (1997) Mol. Endocrinol. 11, of MCF-7 human breast cancer cells. Although Sp1 is impor- 1569 –1580 tant for basal transcription of genes involved in cell growth, 12. Pipao ´ n, C., Tsai, S. Y., and Tsai, M. J. (1999) Mol. Cell. Biol. 19, 2734 –2745 13. Suzuki, Y., Shimada, J., Shudo, K., Matsumura, M., Crippa, M. P., and expression of several cell cycle-regulated genes such as dihy- Kojima, S. (1999) Blood 93, 4264 – 4276 drofolate reductase and hypoxanthine/guanine phosphoribosyl 14. Owen, G. I., Richer, J. K., Tung, L., Takimoto, G., and Horwitz, K. B. (1998) transferase were unaffected in gestation day 8.5-day-old em- J. Biol. Chem. 273, 10696 –10701 15. Lu, S., Jenster, G., and Epner, D. E. (2000) Mol. Endocrinol. 14, 753–760 bryos (5). In contrast, transfection of GC-rich Sp1 oligonucleo- 16. Liu, Z., and Simpson, E. R. (1999) Mol. Cell. Endocrinol. 153, 183–196 tide decoys into A549 human lung adenocarcinoma and U251 17. Sugawara, T., Saito, M., and Fujimoto, S. (2000) Endocrinology 141, 2895–2903 human glioblastoma cells blocked expression of several genes 18. Curtin, D., Jenkins, S., Farmer, N., Anderson, A. C., Haisenleder, D. J., with GC-rich promoters and suppressed cell growth. This ap- Rissman, E., Wilson, E. M., and Shupnik, M. A. (2001) Mol. Endocrinol. 15, proach and others that target GC-rich sequences suggest that 1906 –1917 19. Shimada, J., Suzuki, Y., Kim, S. J., Wang, P. C., Matsumura, M., and Kojima, Sp1 protein may play an important role in cell growth (45, 46); S. (2001) Mol. Endocrinol. 15, 1677–1692 however, these techniques lack specificity because multiple Sp 20. Wang, F., Hoivik, D., Pollenz, R., and Safe, S. (1998) Nucleic Acids Res. 26, 3044 –3052 family proteins bind GC-rich motifs that may influence the 21. Wang, W., Dong, L., Saville, B., and Safe, S. (1999) Mol. Endocrinol. 13, function of other DNA-bound transcription factors. Research in 1373–1387 this laboratory has identified E2-responsive GC-rich motifs in 22. Xie, W., Duan, R., and Safe, S. (1999) Endocrinology 140, 219 –227 23. Sun, G., Porter, W., and Safe, S. (1998) Mol. Endocrinol. 12, 882– 890 promoters of several genes involved in cell proliferation, and 24. Qin, C., Singh, P., and Safe, S. (1999) Endocrinology 140, 2501–2508 these include cyclin D1, thymidylate synthase, c-fos, E2F1, 25. Duan, R., Porter, W., and Safe, S. (1998) Endocrinology 139, 1981–1990 26. Dong, L., Wang, W., Wang, F., Stoner, M., Reed, J. C., Harigai, M., Kladde, M., bcl2, and DNA polymerase  (20 –31). Several approaches were Vyhlidal, C., and Safe, S. (1999) J. Biol. Chem. 174, 32099 –32107 previously used to demonstrate the role of ER/Sp1 as a tran- 27. Xie, W., Duan, R., Chen, I., Samudio, I., and Safe, S. (2000) Endocrinology 141, scription factor complex, and this study was designed to further 2439 –2449 28. Castro-Rivera, E., Samudio, I., and Safe, S. (2001) J. Biol. Chem. 276, investigate this non-classical mechanism of estrogen action 30853–30861 and its involvement in hormone-induced transactivation and 29. Samudio, I., Vyhlidal, C., Wang, F., Stoner, M., Chen, I., Kladde, M., Barhoumi, R., Burghardt, R., and Safe, S. (2001) Endocrinology 142, cell proliferation. The results in Figs. 1–3 clearly demonstrate 1000 –1008 that transfected iSp1 was highly effective for decreasing ex- 30. Saville, B., Wormke, M., Wang, F., Nguyen, T., Enmark, E., Kuiper, G., pression of Sp1 protein in nuclear extracts, and, not surpris- Gustafsson, J.-A., and Safe, S. (2000) J. Biol. Chem. 275, 5379 –5387 28822 Inhibitory RNA for Sp1 Inhibits MCF-7 Cell Proliferation 31. Safe, S. (2001) Vitam. Horm. 62, 231–252 40. Tuschl, T. (2001) Chem. Biochem. 2, 239 –245 32. Petz, L. N., and Nardulli, A. M. (2000) Mol. Endocrinol. 14, 972–985 41. Hammond, S. M., Caudy, A. A., and Hannon, G. J. (2001) Nat. Rev. Genet. 2, 33. Salvatori, L., Ravenna, L., Felli, M. P., Cardillo, M. R., Russo, M. A., Frati, L., 110 –119 Gulino, A., and Petrangeli, E. (2000) Endocrinology 141, 2266 –2274 42. Harborth, J., Elbashir, S. M., Bechert, K., Tuschl, T., and Weber, K. (2001) 34. Kyo, S., Takakura, M., Kanaya, T., Zhuo, W., Fujimoto, K., Nishio, Y., Orimo, J. Cell Sci. 114, 4557– 4565 A., and Inoue, M. (1999) Cancer Res. 59, 5917–5921 43. Foster, J. S., and Wimalasena, J. (1996) Mol. Endocrinol. 10, 488 – 498 35. Tanaka, N., Yonekura, H., Yamagishi, S., Fujimori, H., Yamamoto, Y., and 44. Prall, O. W. J., Sarcevic, B., Musgrove, E. A., Watts, C. K. W., and Sutherland, Yamamoto, H. (2000) J. Biol. Chem. 275, 25781–25790 R. L. (1997) J. Biol. Chem. 272, 10882–10894 36. Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, 45. Ishibashi, H., Nakagawa, K., Onimaru, M., Castellanous, E. J., Kaneda, Y., T. (2001) Nature 411, 494 – 498 Nakashima, Y., Shirasuna, K., and Sueishi, K. (2000) Cancer Res. 60, 37. Fire, A. (1999) Trends. Genet. 15, 358 –363 6531– 6536 38. Sharp, P. A. (2001) Genes Dev. 15, 485– 490 46. Martin, B., Vaquero, A., Priebe, W., and Portugal, J. (1999) Nucleic Acids Res. 39. Paddison, P. J., Caudy, A. A., and Hannon, G. J. (2002) Proc. Natl. Acad. Sci. 27, 3402–3409 U. S. A. 99, 1443–1448

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Journal of Biological ChemistryUnpaywall

Published: Aug 1, 2002

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