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J. Biol. Chem., Vol. 277, Issue 32, 28815-28822, August 9, 2002

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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^*

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

Received for publication, April 19, 2002, and in revised form, May 24, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Small interfering RNA duplexes containing 21-22 nucleotides that mediate sequence-specific mRNA degradation and inhibitory RNA (iRNA) for Sp1 mRNA were used in this study to investigate the role of Sp1 on basal and hormone-induced growth and transactivation in MCF-7 and ZR-75 human breast cancer cells. Transfection of Sp1 iRNA in MCF-7 or ZR-75 cells for 36-44 h decreased Sp1 protein (50-70%) in nuclear extracts, and immunohistochemical analysis showed that the Sp1 protein in transfected MCF-7 cells was barely detectable. In cell cycle progression studies in MCF-7 cells, decreased Sp1 protein was accompanied by a decrease in cells in the S phase and an increase in cells in G₀/G₁, and estrogen-induced G₀/G₁  S phase progression was inhibited in cells treated with iRNA for Sp1. Sp1 iRNA also specifically blocked basal and estrogen-induced transactivation in cells transfected with a GC-rich construct linked to a luciferase reporter gene (pSp1₃), and this was accompanied by decreased Sp1 binding to this GC-rich promoter as determined in gel mobility shift and chromatin immunoprecipitation assays. These results clearly demonstrate the key role of the Sp1 protein in basal and estrogen-induced growth and gene expression in breast cancer cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Sp1 is a member of the Sp and Krüppel-like family of transcription factors that bind GC and CACCC boxes to regulate gene expression (1-3). Sp1 is widely expressed in multiple tissues (4), and targeted disruption of Sp1 in mice results in retarded development and embryo-lethality (5). Sp1 interacts with GC-rich Sp1 binding sites in multiple promoters to regulate gene expression, and there are an increasing number of studies showing that Sp1 interacts with other nuclear proteins, including promoter-bound transcription factors, to attenuate tissue-specific expression of selected genes (1-3). For example, Sp1 and NFY cooperatively interact to regulate multiple genes through NFY-GC-rich motifs, and both proteins also physically interact (6-10). Sp1 also binds estrogen receptor  (ER)¹ and other members of the nuclear receptor family of transcription factors (11-19), and research in our laboratory has focused on the molecular mechanisms of the ligand-dependent activation of ER/Sp1 in breast and endometrial cancer cell lines (20-31). Promoter analysis studies in breast cancer cells have identified GC-rich sites required for hormone activation of several genes including E2F1, DNA polymerase , cyclin D1, insulin-like growth factor-binding protein 4, retinoic acid receptor 1, cathepsin D, vascular endothelial growth factor, c-fos, heat shock protein 27, bcl-2, thymidylate synthase, and adenosine deaminase (20-27, 29-31). Studies in other cell lines have also demonstrated a role for ER/Sp1 activation of the progesterone receptor, epidermal growth factor receptor, telomerase, and receptor for advanced glycation end products (32-35). Activation of ER/Sp1 does not require the DNA binding domain of ER (promoter DNA-independent) and is primarily dependent on the activation function-1 (AF1) of ER (30), whereas DNA-dependent activation through ER binding to estrogen response elements (EREs) is primarily dependent on AF2 of ER.

Recent studies have demonstrated that RNA interference through small inhibitory RNAs (iRNAs) targeted to endogenous or heterologous genes can be used to suppress intracellular expression of these genes in mammalian cells, and this technique is well suited for mechanistic studies on gene/protein function (36-42). This study investigates the role of Sp1 protein in mediating hormone-responsiveness in MCF-7 cells using sequence-specific duplexes of 21 nucleotides targeted to Sp1 mRNA as well as Lamin B1 and the heterologous firefly luciferase gene (GL2) mRNAs. Transfection of iRNA for Sp1 (iSp1) decreases (40-60%) the expression of nuclear Sp1 protein in ER-positive MCF-7 and ZR-75 human breast cancer cell extracts. In transfected cells, Sp1 protein is barely detectable by immunofluorescence, and both basal and estrogen-inducible transactivation is decreased in cells transfected with iSp1 and a GC-rich construct. These data, combined with results showing that iSp1 inhibits hormone-induced MCF-7 cell cycle progression from G₀/G₁ to S phase, demonstrate that ER/Sp1-mediated transactivation plays a major role in ER-positive breast cancer cell growth.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Lines-- MCF-7 and ZR-75 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). DME/F12 with and without phenol red, 100× antibiotic/antimycotic solution, propidium idodide, and E2 were purchased from Sigma. Fetal bovine serum was purchased from Intergen (Purchase, NY). [-³²P]ATP (300Ci/mmol) was obtained from PerkinElmer Life Sciences. Poly(dI-dC) and T4 polynucleotide kinase were purchased from Roche Molecular Biochemicals (Indianapolis, IN). Antibodies for proteins Sp1, Lamin B1, and ER proteins were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Human ER expression plasmid was provided by Dr. Ming-Jer Tsai, Baylor College of Medicine (Houston, TX). The pSp1₃ construct contains three consensus Sp1 binding sites and the pERE₃ construct contains three EREs. The oligonucleotides were linked to the bacterial luciferase gene and cloned into BamHI-HindIII-cut XP-2 plasmid obtained from ATCC. Lysis buffer, luciferase reagent, and RNase were obtained from Promega Corp. (Madision, WI). All other chemicals and biochemicals were of the highest quality available from commercial sources. iRNAs were prepared by IDT (Coralville, IA) and targeted the coding region 153-173, 672-694, and 1811-1833 relative to the start codon of GL2, Lamin B1 (LMN), and Sp1 genes, respectively. Single-stranded RNAs were annealed by incubating 20 µM of each strand in annealing buffer (100 mM potassium acetate, 30 mM HEPES buffer at pH 7.4, 2 mM magnesium acetate) for 1 min at 90 °C followed by 1 h at 37 °C. The iRNA duplexes used in this study are indicated as follows:

Transfection of MCF-7 and ZR-75 Cells and Preparation of Nuclear Extracts-- Cells were cultured in 6-well plates in 2 ml of DME/F12 medium supplemented with 5% fetal bovine serum. After 16-20 h when cells were 50-60% confluent, iRNA duplexes and/or reporter gene constructs were transfected using LipofectAMINE Plus Reagent (Invitrogen). The effects of iSp1 on hormone-induced transactivation was investigated in MCF-7 cells treated with 10 nM E2 and cotransfected with pSp1₃ (500 ng) or pERE₃ (500 ng) and ER expression plasmid (200 ng). Based on the results of preliminary studies, 0.75 µg of iRNA duplex was transfected in each well to give a final concentration of 50 nM. Cells were harvested 36-44 h after transfection by manual scraping in 1× lysis buffer (Promega). For whole cell extracts, cells were frozen in liquid nitrogen for 30 s, vortexed for 30 s, and centrifuged at 12,000 × g for 1 min. Lysates were assayed for luciferase activity using luciferase assay reagent (Promega). -Galactosidase activity was measured using Tropix Galacto-Light Plus assay system (Tropix, Bedford, MA) in a Lumicount microwell plate reader (Packard Instrument Co.). For nuclear extracts, cells were washed in PBS (2×), scraped in 1 ml of 1× lysis buffer, incubated at 4 °C for 15 min, and centrifuged at 14,000 × 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 added to the cell pellet and incubated for 45 min at 4 °C with frequent vortexing. Nuclei were pelleted by centrifugation at 14,000 × g for 1 min at 4 °C, and aliquots of supernatant were stored at 80 °C and used for Western blot analysis and gel shift assays.

Western Immunoblot-- An aliquot of nuclear protein (30 µg) was diluted with loading buffer, boiled, and loaded on a 7.5% SDS-polyacrylamide gel. Samples were electrophoresed at 150-180 V for 3-4 h, and separated proteins were transferred to polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) in buffer containing 48 mM Tris-HCl, 29 mM glycine, and 0.025% SDS. Proteins were detected by incubation with polyclonal primary antibodies Sp1-PEP2, Lamin B1-C20, and ER-G20 (all 1:1000 dilution) against Sp1, Lamin B1, and ER proteins, respectively, followed by blotting with horseradish peroxidase-conjugated anti-rabbit (for Sp1 and ER) or anti-goat (for Lamin B) secondary antibody (1:5000 dilution). Blots were then exposed to chemiluminescent substrate (PerkinElmer Life Sciences) and placed in Kodak X-Omat AR autoradiography film. Band intensities were determined by a scanning laser densitometer (Sharp Electronics Corp., Mahwah, NJ) using Zero-D Scanalytics software (Scanalytics Corp., Billerica, MA).

FACS Analysis-- Cells were transfected with iRNAs for Sp1 or GL2 and, after 20-24 h, treated with Me₂SO or 20 nM E2 for 18-20 h in serum-free medium. Cells were then trypsinized and ~2 × 10⁶ cells were centrifuged and resuspended after removal of trypsin in 1 ml of staining solution containing 50 µg/ml propidium iodide, 4 mM sodium citrate, 30 units/ml RNase, and 0.1% Triton X-100, pH 7.8. Cells were incubated at 37 °C for 10 min, and then prior to FACS analysis sodium chloride was added to give a final concentration of 0.15 M. Cells were analyzed on a FACSCalibur flow cytometer (BD PharMingen) using CellQuest (BD PharMingen) acquisition software. Propidium iodide fluorescence was collected through a 585/42-nm bandpass filter, and list mode data were acquired on a minimum of 12,000 single cells defined by a dot plot of PI-width versus PI-area. Data analysis was performed in ModFit LT (Verity Software House, Topsham, ME) using PI-width versus PI-area to exclude cell aggregates. FlowJo (Treestar, Inc., Palo Alto, CA) was used to generate plots shown in the figures.

Electrophoretic Mobility Shift Assay (EMSA)-- Consensus Sp1 oligonucleotide (28, 30) was synthesized and annealed, and 5-pmol aliquots were 5'-end-labeled using T4 kinase and [-³²P]ATP. A 30-µl EMSA reaction mixture contained ~100 mM KCl, 3 µg of crude nuclear protein, 1 µg poly(dI-dC), with or without unlabeled competitor oligonucleotide, and 10 fmol of radiolabeled probe. After incubation for 20 min on ice, antibodies against Sp1 protein were added and incubated another 20 min on ice. Protein-DNA complexes were resolved by 5% polyacrylamide gel electrophoresis in 1× Tris borate/EDTA buffer (0.09 M Tris-base, 0.09 M boric acid, 2 mM EDTA, pH 8.3) at 120 V at 4 °C for 2-3 h. Specific DNA-protein and antibody-supershifted complexes were observed as retarded bands in the gel.

Immunocytochemistry-- MCF-7 cells were seeded in Lab-Tek Chamber slides (Nalge Nunc International, Naperville, IL) at 100,000 cells/well in DME/F12 medium supplemented with 5% fetal bovine serum, and after 14 h cells were transferred into serum-free medium for 8-10 h. Cells were then transfected with iRNAs, and after 36-44 h the media chamber was detached and the remaining glass slides were washed in Dulbecco's PBS. After washing, the glass slides were fixed with cold (20 °C) methanol for 10 min, and then slides were washed in 0.3% PBS/Tween for 5 min (2×) before blocking with 5% rabbit or goat serum in antibody dilution buffer (stock solution: 100 ml of PBS/Tween, 1 g of bovine serum albumin, 45 ml of glycerol, pH 8.0) for 1 h at 20 °C. After removal of the blocking solution, rabbit Sp1-PEP2 or goat Lamin B1 polyclonal antibodies were added in antibody dilution buffer (1:200) and incubated for 12 h at 4 °C. Slides were washed for 10 min with 0.3% Tween in 0.02 M PBS (3×) and incubated with fluorescein isothiocyanate-conjugated anti-rabbit or anti-goat secondary antibodies (1:1000 dilution) for 2 h at 20 °C. Slides were then washed for 10 min in 0.3% PBS-Tween (4×). Slides were mounted in ProLonged antifading medium (Molecular Probes, Inc., Eugene, OR), and cover slips were sealed using Nailslicks nail polish (Noxell Corp., Hunt Valley, MD). Fluorescence imaging was performed using Carlzeiss Axiophoto 2 (Calzeiss, Inc., Thornwood, NY). Images were captured using Adobe Photoshop 5.5 using identical camera settings.

Chromatin Immunoprecipitation Assay (ChIP)-- Cells were transfected with iSp1 or iGL2 for 36 h, then treated with Me₂SO. MCF-7 cells were then collected, suspended in 1× PBS with 1 mM phenylmethylsulfonyl fluoride, and formaldehyde was added to the medium to give a 1% solution that was incubated with shaking for 10 min at 20 °C. Glycine was then added (0.125 M) and, after further incubation for 10 min, cells were collected by centrifugation and washed with PBS and 1 nM phenylmethylsulfonyl fluoride. Cells were then resuspended in swell buffer (85 mM potassium chloride, 0.5% Nonidet P-40, 1 nM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, and aprotinin at pH 8.0), homogenized, and nuclei were isolated by centrifugation at 1500 × g for 30 s. Nuclei were then resuspended in sonication buffer (1% SDS, 10 nM EDTA, 50 mM Tris at pH 8.1) and sonicated for 45-60 s to obtain chromatin with appropriate fragment lengths (500-1000 bp). The sonicated extract was then centrifuged at 15,000 × g for 10 min at 0 °C, aliquoted, and stored at 70 °C until used. The cross-linked chromatin preparations were diluted in buffer (1% Triton X, 100 mM sodium chloride, 0.5% SDS, 5 mM EDTA and Tris at pH 8.1), and 20 µl of ultralink protein A or G or A/G beads were added per 100 µl of chromatin, and incubated for 4 h at 4 °C. Beads were collected by centrifugation, and salmon sperm DNA, specific antibodies, and 20 µl of ultralink beads were added to the supernatant. The mixture was incubated for 6 h at 4 °C. An aliquot was treated at 65 °C to reverse the cross-links, extracted with phenol/chloroform, and DNA was precipitated with ethanol. This aliquot was used as an input control. Immunoprecipitated samples were then centrifuged. Beads were resuspended in dialysis buffer, vortexed for 5 min at 20 °C, and centrifuged at 15,000 × g for 10 s. Beads were then resuspended in immunoprecipitation buffer (11 mM Tris, 500 mM lithium chloride, 1% Nonidet P-40, 1% deoxycholic acid at pH 8,0) and vortexed for 5 min at 20 °C. Procedures with the dialysis and immunoprecipitation buffers were repeated (3-4 times), and beads were then resuspended in elution buffer (50 nM NaHCO₃, 1% SDS, 1.5 µg/ml sonicated salmon sperm DNA), vortexed, and incubated at 65 °C for 15 min. Supernatants were then isolated by centrifugation and incubated at 65 °C for 6 h to reverse protein-DNA cross-links. Wizard PCR kits (Promega) were used for additional DNA cleanup. A portion of the purified immunoprecipitated DNA and 0.2% of the input control were used for [-P³²]dCTP incorporation PCR. One-fourth of a microliter of [-P³²]dCTP (3000 Ci/mmol) was added to a 25-µl PCR reaction (3% Me₂SO), 1 M betaine, and 1.5 mM magnesium chloride) and subjected to one cycle of 95 °C × 5 min, 5 cycles of 95 °C × 30 s, 60 °C × 30 s, 5 cycles of 95 °C × 30 s, 55 °C × 30 s, 72 °C × 30 s, and 5 cycles of 95 °C × 30 s, 48 °C × 30 s, 72 °C × 30 s, followed by one cycle at 72 °C for 4 min. Reactions were loaded on a 10-15% non-denaturing acrylamide gel. The gel was then dried and exposed to a phosphor screen for 24 h. The primers used for PCR of the GC-rich region of pSp1₃ are indicated as follows: pxp2-luc-Fw (6128), 5'-GTTTGTCCAAACTCATCAATG-3'; Rv (105), 5'-CTTTATGTTTTTGGCGTCTTC-3'.

Statistical Analysis-- Statistical significance was determined by analysis of variance and Scheffe's test, and the levels of probability are noted. The results are expressed as means ± S.D. for at least three separate (replicate) experiments for each treatment.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

iSp1 Specifically Decreases Nuclear Sp1 Protein Levels in ER-positive Human Breast Cancer Cells-- Results of preliminary studies indicate that iSp1 and iLMN were most effective at decreasing cellular protein levels by treating cells for 36-44 h with 0.75 µg of the duplex oligonucleotides. The results illustrated in Fig. 1, A and B show that transfection of iSp1 in MCF-7 cells significantly decreased Sp1 protein by ~60% in nuclear extracts, whereas immunoreactive Lamin B1 and ER levels were unchanged. In contrast, transfection of iLMN decreased Lamin B1 but not Sp1 or ER protein levels, thus demonstrating the specificity of the iRNAs. The results summarized in Fig. 1C confirm that iSp1 (but not iLMN) also significantly decreased Sp1 protein in ER-positive ZR-75 cells. The effects of iRNAs on nuclear protein levels were also investigated in gel mobility shift assays using extracts from MCF-7 or ZR-75 cells (Fig. 2, A and B) and a consensus GC-rich oligonucleotide [³²P]Sp1 that binds Sp1 and other Sp1 family proteins. Incubation of nuclear extracts from MCF-7 cells with [³²P]Sp1 gave a profile of retarded bands (lane 2) associated with Sp1- and Sp3-DNA complexes (28). The intensity of the former complex was decreased after incubation with unlabeled Sp1 oligonucleotide (lane 5) and supershifted with Sp1 antibodies (lane 6). In cells transfected with iSp1, there was a decrease in retarded band intensity (lane 4), whereas iLMN did not affect retarded band intensities. The results obtained for ZR-75 cells (Fig. 2B) were similar to those observed in MCF-7 cells and confirm the effectiveness and specificity of iSp1 for selectively decreasing Sp1 protein in breast cancer cells.

View larger version (42K): [in this window] [in a new window]   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.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Binding of [32P]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 [32P]Sp1 was determined in gel mobility shift assays as described under "Materials and Methods." C, chromatin immunoprecipitation assay. MCF-7 cells were transfected with pSp13 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."

We have also used a chromatin immunoprecipitation assay to further investigate the in situ effects of iSp1 on Sp1-DNA interactions. MCF-7 cells were cotransfected with iSp1 or iGL2 and a construct containing three tandem GC-rich Sp1 binding sites (pSp1₃), and after 36-44 h, cells were treated with formaldehyde to cross-link DNA-bound proteins. After immunoprecipitation with Sp1 or Sp3 antibodies and removal of the cross-links, PCR was used to identify the GC-rich region of pSp1₃ as part of the immunoprecipitable complexes. The results showed that iSp1 decreased interaction of Sp1 with the GC-rich promoter compared with that observed in cells transfected with iGL2, whereas the intensity of PCR products was similar for Sp3 immunoprecipitable complexes. Thus, results of Western blots, gel mobility shift, and ChIP demonstrate a significant (40-60%) decrease in Sp1 protein in breast cancer cells transfected with iSp1.

Sp1 Protein Expression, Sp1, and ER/Sp1-mediated Transactivation in MCF-7 Cells Transfected with iSp1-- Transfection with LipofectAMINE results in >40-60% transfection efficiency in MCF-7 cells, suggesting that iSp1 is highly effective in decreasing Sp1 expression in transfected cells. This was further investigated in MCF-7 cells by immunofluorescence analysis of Sp1 or lamin protein in MCF-7 transfected with iSp1 or iLMN (Fig. 3). Panels A and E are control panels where the primary antibody for lamin (panel A) or Sp1 (panel E) has been omitted. Panel C is a control for lamin (iLMN) showing immunofluorescence of Lamin B and phase contrast (panel B). In cells transfected with iLMN, most of the cells exhibited either significantly decreased Lamin B expression (transfected cells) or lamin expression was unchanged (non-transfected cells). Sp1 staining was observed in untreated MCF-7 cells (panel F) or in cells transfected with iLMN (panel G); however, in cells transfected with iSp1, there was a marked decrease of Sp1 staining in most cells, whereas the non-transfected cells were essentially unchanged. These data demonstrate that transfected iSp1 but not iLMN were highly effective in decreasing cellular expression of Sp1, and this accounts for the decreases in Sp1 protein in nuclear extracts (Figs. 1 and 2).

View larger version (84K): [in this window] [in a new window]   Fig. 3.   Immunoflourescence of Sp1 and Lamin B in MCF-7 cells transfected with iSp1 and iLMN. MCF-7 cells were untreated (A and E), transfected with iSp1 (H), iLMN (D and G), and stained with Sp1 (F-H) or Lamin B (B-D) antibodies. Immunofluorescence was determined as described under "Materials and Methods."

The results in Fig. 4A summarize the effects of iLMN, iGL2, and iSp1 on luciferase activity in MCF-7 cells transfected with pSp1₃ and the iRNAs. iLMN did not significantly decrease activity, whereas iGL2 (which is targeted to the luciferase mRNA) and iSp1 both inhibited luciferase activity. In this study (Fig. 4, A and B), there was a >60-77% decrease in basal activity in cells transfected with iSp1. Moreover, E2 induced luciferase activity in MCF-7 cells transfected with pSp1₃ as previously described (28), and in cells cotransfected with iSp1 there was a >80% decrease in hormone-induced transactivation. Thus, iSp1 inhibited both basal and E2 induced luciferase activity in MCF-7 cells transfected with pSp1₃. In contrast, hormone-induced transactivation in MCF-7 cells transfected with pERE₃ was not affected by cotransfection with iLMN or iSp1, whereas iGL2 decreased activity in cells treated with Me₂SO or E2 (Fig. 4C). Thus, iSp1 specifically blocks hormone-induced transactivation in cells transfected with pSp1₃ but not pERE₃.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Effects of iLMN, iSp1, and iGL2 on luciferase activity in MCF-7 cells transfected with pSp13 and treated with Me2SO or E2. A, effects of inhibitor RNAs on basal activity. MCF-7 cells were transfected with pSp13 alone or in combination with iLMN, iGL2, or iSp1 and treated with Me2SO. Luciferase activity was determined as described under "Materials and Methods." B, iSp1-mediated inhibition of transactivation in cells transfected with pSp13. Cells were transfected with pSp13 and iSp1, treated with Me2SO or 10 nM E2, and luciferase activity was determined as described under "Materials and Methods." C, effects of iSp1 on cells transfected with pERE3. Cells were transfected with pERE3 and iLMN, iGL2, or iSp1 treated with Me2SO 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 replicate determinations for each treatment group, and significant (p < 0.05) decreases in activity are indicated by an asterisk.

iSp1 Inhibits Hormone-induced MCF-7 Cell Cycle Progression-- Promoter regions in several genes associated with cell proliferation contain E2-responsive GC-rich motifs (20-31); however, the role of ER/Sp1 in mediating cell growth can only be inferred from these studies. The role of Sp1 in hormone-induced cell cycle progression was further investigated to determine the effects of iSp1 and iGL2 (a control) on distribution of MCF-7 cells in G₀/G₁, G₂-M, and S phases after treatment with solvent (Me₂SO) or 20 nM E2 for 18-20 h. At this time point, iRNA for Sp1 increased the percent of solvent-treated cells in G₀/G₁ from 75.3 to 78.3% and decreased the percent in S phase (from 15.1 to 12.1). In a parallel study in untreated cells at an earlier time point (8-10 h), a 5% decrease in cells in S phase and a similar increase in cells in G₀/G₁ was observed (data not shown). More dramatic changes were observed for the effects of iSp1 on E2-induced proliferation of MCF-7 cells. For example, in cells treated with Me₂SO or 20 nM E2, the percent of cells in G₀/G₁:S phase was 75.3:9.57% or 66.1:23.7%, respectively, showing a dramatic increase in G₀/G₁  S progression after treatment with E2, and this has been observed in other studies (43, 44). In contrast, the percent of cells in G₀/G₁:S phase in cells treated with iSp1 was 71.9:17.3%, indicating that hormone-induced cell cycle progression was markedly decreased by ablating cellular expression of Sp1 protein, whereas transfection of the control iGL2 did not affect cell cycle progression. These results demonstrate for the first time that Sp1 protein and ER/Sp1-mediated transactivation are important for hormone-induced proliferation of MCF-7 cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The development of genetic technologies to regulate or delete expression of endogenous genes has been extensively used to probe the role of specific genes on biological function. For example, the generation of knock-out/knock-in mice and the overexpression of genes in transgenic animal models has provided unique insights on gene function in normal physiology and disease processes. RNA interference by double-stranded RNA involves the sequence-specific post-transcriptional silencing of genes that has been widely described and used in plants and animals (37, 38, 40, 41). It has recently been shown that small interfering RNA duplexes (21-25 nucleotides) targeted to specific genes can now be introduced into mammalian cells in culture to decrease RNA/protein expression (36-42). Elbashir et al. (36) recently reported iRNA duplexes for endogenous and exogenous genes decreased their corresponding protein and/or protein-dependent activities in several mammalian cell lines including NIH 3T3, HeLa, COS-7, and 293 cells.

This study has used the iRNA approach for investigating the role of Sp1 protein in the growth and hormone-responsiveness of MCF-7 human breast cancer cells. Although Sp1 is important for basal transcription of genes involved in cell growth, expression of several cell cycle-regulated genes such as dihydrofolate reductase and hypoxanthine/guanine phosphoribosyl transferase were unaffected in gestation day 8.5-day-old embryos (5). In contrast, transfection of GC-rich Sp1 oligonucleotide decoys into A549 human lung adenocarcinoma and U251 human glioblastoma cells blocked expression of several genes with GC-rich promoters and suppressed cell growth. This approach and others that target GC-rich sequences suggest that Sp1 protein may play an important role in cell growth (45, 46); however, these techniques lack specificity because multiple Sp family proteins bind GC-rich motifs that may influence the function of other DNA-bound transcription factors. Research in this laboratory has identified E2-responsive GC-rich motifs in promoters of several genes involved in cell proliferation, and these include cyclin D1, thymidylate synthase, c-fos, E2F1, bcl2, and DNA polymerase  (20-31). Several approaches were previously used to demonstrate the role of ER/Sp1 as a transcription factor complex, and this study was designed to further investigate this non-classical mechanism of estrogen action and its involvement in hormone-induced transactivation and cell proliferation. The results in Figs. 1-3 clearly demonstrate that transfected iSp1 was highly effective for decreasing expression of Sp1 protein in nuclear extracts, and, not surprisingly, immunofluorescence studies indicate that Sp1 protein is barely detectable in transfected cells (Fig. 3). The high efficiency of iSp1 for ablating Sp1 protein in transfected cells was observed in MCF-7 cells cotransfected with iSp1 and pSp1₃, an E2-responsive GC-rich construct that serves as a surrogate for other GC-rich E2-responsive gene promoters (Fig. 4). In these transfection studies, iSp1 significantly decreased both basal and E2-induced luciferase activities confirming the role of ER/Sp1 in ligand-activated transcription.

Treatment of growth-arrested MCF-7 cells with E2 results in cell cycle progression that is characterized by a decrease in cells in G₀/G₁ and an increase in cells in S phase (43, 44) (Fig. 5). In untreated cells, iSp1 further increased the percent of cells in G₀/G₁ (from 75.3 to 78.3%) and decreased the number of cell in S phase (from 15.1 to 12.1%). Because FACS analysis was carried out on the total cell population (transfected and non-transfected), the response of MCF-7 cells to transfected iSp1 demonstrates the important role of Sp1-regulated genes in basal growth of these cells. The effects of iSp1 were more dramatic in reversing hormone-induced cell cycle progression and blocking a high proportion of these cells from progression to S phase. These data are consistent with the results of previous studies showing that cyclin D1 and other genes important for cell proliferation are regulated by ER/Sp1 (21, 25, 26, 28, 29). Future studies will use iRNAs to further investigate the role of Sp1, other Sp-like proteins, and coregulatory factors on the growth of MCF-7 and other hormone-dependent cell lines and to identify key genes that are integral for these responses.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Effects of iSp1 on hormone-induced cell cycle progression in MCF-7 cells. Serum-starved MCF-7 cells were treated with Me2SO or E2 alone or cotransfected with iGL2 and iSp1, and the percent of distribution of cells in G1/G0, S, and G2/M were determined by FACS analysis as described under "Materials and Methods." Similar results were observed in a duplicate analysis.

	FOOTNOTES

^* 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 article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Veterinary Physiology and Pharmacology, Texas A&M University, 4466 TAMU, Vet. Res. Bldg. 409, College Station, TX 77843-4466. Tel.: 979-845-5988; Fax: 979-862-4929; E-mail: ssafe@cvm.tamu.edu.

Published, JBC Papers in Press, June 6, 2002, DOI 10.1074/jbc.M203828200

	ABBREVIATIONS

The abbreviations used are: ER, estrogen receptor ; ERE, estrogen response elements; i, inhibitory; FACS, fluorescence-activated cell sorter; PI, propidium idodide; EMSA, electrophoretic mobility shift assay; PBS, phosphate-buffered saline, ChIP, chromatin immunoprecipitation assay; LMN, Lamin B1.

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