Indole-3-carbinol Inhibits CDK6 Expression in Human MCF-7 Breast Cancer Cells by Disrupting Sp1 Transcription Factor Interactions with a Composite Element in the CDK6 Gene Promoter*

Erin J. CramDagger §, Betty D. LiuDagger , Leonard F. Bjeldanes, and Gary L. FirestoneDagger ||

From the Dagger  Department of Molecular and Cell Biology, the Cancer Research Laboratory, and the  Department of Nutrition and Toxicology, University of California, Berkeley, California 94720

Received for publication, November 21, 2000, and in revised form, March 27, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Indole-3-carbinol (I3C), a compound naturally occurring in Brassica vegetables, can induce a G1 cell cycle arrest of human MCF-7 breast cancer cells that is accompanied by the selective inhibition of cyclin-dependent kinase 6 (CDK6) expression. Reverse transcriptase-polymerase chain reaction analysis of CDK6 mRNA decay rates revealed that I3C had no effect on CDK6 transcript stability. We report the first identification and functional characterization of the CDK6 promoter in order to determine whether I3C inhibits CDK6 transcription. In MCF-7 cells stably transfected with CDK6 promoter-linked luciferase reporter plasmids, I3C inhibited CDK6 promoter activity in an I3C-specific response that was not a consequence of the growth-arrested state of the cells. Deletion analysis revealed a 167-base pair I3C-responsive region of the CDK6 promoter between -805 and -638. Site-specific mutations within this region revealed that both Sp1 and Ets-like sites, which are spaced 5 base pairs apart, were necessary for I3C responsiveness in the context of the CDK6 promoter. Electrophoretic mobility shift analysis of protein-DNA complexes formed with nuclear proteins isolated from I3C-treated and -untreated cells, in combination with supershift assays using Sp1 antibodies, demonstrated that the Sp1-binding site in the CDK6 promoter forms a specific I3C-responsive DNA-protein complex that contains the Sp1 transcription factor. Taken together, our results suggest that I3C down-regulates CDK6 transcription by targeting Sp1 at a composite DNA site in the CDK6 promoter.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Indole-3-carbinol (I3C),1 a naturally occurring component of Brassica vegetables such as cabbage, broccoli, and brussels sprouts, is a promising chemopreventative agent that has been shown to be effective in short term assays relevant to carcinogen-induced DNA damage, oxidative stress, and tumor initiation and promotion (1). Many studies have demonstrated that exposure to dietary I3C markedly reduces the incidence and multiplicity of spontaneous and carcinogen-induced mammary tumors with low levels of toxicity (2-5). Dietary I3C has anti-estrogenic activity in vivo that has been proposed to account for some of its protective and anti-proliferative effects on mammary tumor formation. When ingested, I3C is converted into a variety of acid-catalyzed oligomers (6) that likely account for many of the long term anti-estrogenic biological activities of dietary I3C (7, 8). In contrast to this dietary pathway, recent evidence has revealed that I3C itself has potent anti-proliferative activity. For example, ectopic application of I3C inhibited skin tumor formation in mouse models (9). In addition, I3C, but not its major acid-catalyzed derivatives, arrests the growth of both estrogen-responsive MCF-7 and estrogen receptor-negative MDA-MB-231 human breast cancer cells by inducing a G1 block in cell cycle progression (10).

Regulated changes in the expression and/or activity of cell cycle components that act within the G1 phase of the cell cycle have been closely associated with the alterations in the proliferation rate of normal and transformed mammary epithelial cells induced by extracellular signals (11-13). Key targets of these pathways are specific sets of cyclin/cyclin-dependent kinase (CDK) protein complexes, which function at specific stages of the cell cycle (14, 15). The timely appearance and degradation of the cyclin-CDK protein complexes drives the cell cycle events in G1 phase of cell cycle (Cyclin D·CDK4, Cyclin D·CDK6, and Cyclin E·CDK2), DNA replication in S phase (Cyclin E·CDK2 and Cyclin A·CDK2), and cell division in G2 and M phases (Cyclin A·CDK1 and Cyclin B·CDK1) (16-18). The activity of the CDK is tightly regulated during passage through the cell cycle by cyclin association, subunit phosphorylation, and interaction with a variety of CDK inhibitors (15). Early in G1, an active Cyclin D·CDK4 or Cyclin D·CDK6 complex phosphorylates retinoblastoma protein family members (19, 20). One critical role for these Cyclin D·CDK complexes in G1 progression may be to sequester p21 and p27 so that Cyclin E·CDK2 can become active and complete the series of events required for progression through S phase. Retinoblastoma becomes hyperphosphorylated late in G1 by Cyclin E·CDK2 resulting in release of the E2F family of transcription factors (21), which activate various genes to modulate progression from G1 into S phase (22, 23).

The loss of normal cell cycle control in G1 has been implicated in mammary tumor development and proliferation (12). For example, up to 45% of human breast cancers show an aberrant expression and/or amplification of Cyclin D1 or Cyclin E (24-27). We have recently demonstrated that the I3C signaling pathway controls the proliferation of human breast cancer cells by targeting specific G1-acting cell cycle components (10, 28). The I3C-mediated growth arrest and G1 block in the cell cycle progression of MCF-7 cells is accompanied by a striking decrease in CDK6 mRNA and protein that accounts for the loss of total cellular CDK6 kinase activity. I3C treatment also results in the inhibition of CDK2-specific enzymatic activity, modest increases in the levels of the p21 and p27 CDK inhibitors, and a concomitant decrease in the phosphorylation of endogenous retinoblastoma protein.

An intriguing feature of the I3C-induced G1 arrest of MCF-7 cells is that I3C treatment represses the production of CDK6 protein and transcripts (10). The enzymatic activities of CDK6, CDK4, and CDK2 are highly regulated at a post-translational or protein complex level (17). There are far fewer examples of cell cycle regulation via the decreased availability of G1 CDKs. For example, staurosporine treatment of MDA-361 breast carcinoma cells (29), herbimycin A treatment of T cells (30), and apoptotic signaling through the IgM surface antigen receptor in B cells (31) all decrease the protein levels of CDK6 and/or CDK4. In only a few instances has the control of CDK mRNA expression been observed. For example, an increase in CDK2 mRNA has been found following stimulation with phorbol ester (32), anti-IgM stimulation (33), or serum in several cell types (34). In addition, DNA damage can induce prolonged down-regulation of CDK1 promoter activity (35).

In order to elucidate further the mechanism by which I3C signaling induces a cell cycle arrest of human breast cancer cells, we have cloned and characterized the human CDK6 promoter. In this study we establish that the I3C treatment down-regulates CDK6 transcription by selectively disrupting the interactions of Sp1 with a composite DNA-binding site within the CDK6 promoter. Our observations represent the first example of the regulation of CDK6 transcription through specific DNA elements that control CDK6 promoter activity and may help to elucidate the pathway by which I3C regulates the cell cycle progression of human breast cancer cells.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Materials-- Dulbecco's modified Eagle's medium, fetal bovine serum, calcium- and magnesium-free phosphate-buffered saline, L-glutamine, and trypsin-EDTA were supplied by BioWhittaker (Walersville, MD). I3C was purchased from Aldrich and recrystallized in hot toluene prior to use. Actinomycin D, insulin (bovine), and tamoxifen ([Z]-1-[p-dimethylaminoethoxyphenyl]-1,2-diphenyl-1-butene) citrate salt were obtained from Sigma. [gamma -32P]ATP (3,000 Ci/mmol) and [3H]acetyl coenzyme A (200 mCi/mmol) were obtained from PerkinElmer Life Sciences. Oligonucleotide primers for PCR were purchased from Integrated Diagnostic Technologies (Coralville, IA). Salts and other chemicals used were of the highest purity available and generally purchased from Sigma.

Cloning of the cdk6 Promoter and Generation of Luciferase Reporter Constructs-- By using a BLAST search of the expressed sequence tag (EST) data base, CDK6 was identified as EST SWSS3105, which had been mapped to chromosome 7q21. This region of the human genome has been cloned into bacterial artificial chromosomes (BAC) and sequenced by the Human Genome Sequencing Project. Sequences for BAC clones covering the 7q21 region were searched for regions of identity with CDK6-coding sequences. BAC H_GS119P05 contained exon 1 of CDK6 and 6000 bp of 5'-sequences, BAC H_DJ0850G01 contained exons 2 and 3, and BAC H_DJ1099C19 contained exons 4-7. BAC H_GS119P05 was purchased from Genome Systems (now Incyte, St. Louis, MO). The GenBankTM accession number for the CDK6 promoter is AF332591.

A 2464-bp fragment containing the CDK6 promoter was amplified using PCR and subcloned into PGL2-basic luciferase expression vectors using MluI and NheI restriction sites engineered into the PCR primers: -2464, 5'-ACGCGTGACGCACGTTTGCTGTAGTTTCC-3', and +24, 5'-GCTAGCCCCGGAGATCGGTCTAGCTTT-3'. BAC DNA was isolated using a Qiagen (Valencia, CA) maxi prep DNA isolation kit, according to the manufacturer's instructions. For PCR amplification, 50 µl of PCRs (1× Tth PCR buffer (Promega, Madison, WI), 1.1 mM Mg(C2H3O2)2, 0.2 mM dNTPs, 1 unit Tth polymerase, 2% Me2SO, 4% glycerol, and 0.2 µM each primer) were amplified for 30 cycles (94 °C, 30 s/62 °C, 1 min/72 °C, 4 min) with a 72 °C, 10-min extender and a 94 °C, 2-min hot start. All sequences were confirmed by automated DNA sequencing. Sequential 5'-deletion constructs were generated by PCR from the -2464-bp luciferase construct. All constructs have a common 3' end with the +24 primer. Forward primers all contain an MluI restriction enzyme site at the 5' end as follows: -920, 5'-ACGCGTAGACCATTTGTGTATGCGAGTCGT-3'; -805, 5'-ACGCGTAGCATCGGGTTACAGG-3'; -638, 5'-ACGCGTGGCGCAACACAATGATATAGGG-3'; -450 5'-ACGCGTCTGCAGGGAAAGAAAAGTAACTTCG-3'; and -196, 5'-ACGCGTGTTGAGGACTTCGCTTCGA-3'.

For PCR amplification 50 µl of PCRs (1× Taq polymerase buffer Mg2+-free, 0.2 mM dNTPs, 1 unit of Taq polymerase (Life Technologies, Inc.), 1.5 mM MgCl2, 0.2 µM each primer) were amplified for 30 cycles (95 °C, 30 s/60 °C, 30 s/70 °C, 30 s). PCR products were gel-purified using Qiagen QIAEXII Gel Extraction Kit, cloned into T-vector, digested with MluI and NheI, and sub-cloned into PGL2-basic, a reporter construct containing no promoter sequences with minimal basal luciferase activity (Promega, Madison, WI).

To generate the transcription factor-binding site mutant fragments, putative binding sites were altered to NcoI restriction enzyme sites by PCR mutagenesis from the -920-bp luciferase construct. For each construct, two overlapping fragments were amplified, cut with the appropriate restriction enzymes, and cloned into PGL2-basic luciferase expression vector. All constructs have a common 3' end with the +24-bp primer and a common 5' end with the -920-bp primer.

Primers used for construction of mutant -920-bp fragments are as follows: Ets mut F, 5'- TCTCCCTGCCATGGCGGGCCTCA-3'; Ets mut R, 5'-TGAGGCTCGCCATGGCAGGGAGAC-3'; Sp1 mut F, 5'-TCAGGAGCCATGGAGGGTAGCGGCGCAA-3'; Sp1 mut R, 5'-CTACCCTCCATGGCTCCTGAGGCCCGGA-3'; NFkappa B mut F, 5'-TCCTGGCCATGGCCACCCACATTCTGC-3'; and NFkappa B mut R, 5'-GGTGGCCATGGCCAGGACCCTGTAACC-3'.

MCF-7 Cell Lines and Methods of Culture-- MCF-7 human breast cancer cell lines were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1.25 ml of 20,000 units/ml penicillin/streptomycin, 2 mM L-glutamine, and 10 µg/ml insulin. Cells were propagated in a 37 °C humidified chamber containing 5% CO2. Cell culture medium was routinely changed every 48 h. I3C was dissolved in dimethyl sulfoxide (Me2SO, 99.9% high pressure liquid chromatography grade, Aldrich) at concentrations 1000-fold higher than the final concentration in the medium. Indole-3-carbinol was added to a final concentration of 200 µM, tryptophol to 200 µM, tamoxifen to 1 µM, and actinomycin D to 1 µg/ml.

Reverse Transcriptase-Polymerase Chain Reaction-- Total RNA from MCF-7 cells treated with Me2SO, I3C, and/or actinomycin D was extracted using the guanidinium thiocyanate method (36), and DNase was treated with RQ1 DNase (Promega, Madison, WI). 2 µg of total RNA was used to synthesize cDNA using Moloney murine leukemia virus-reverse transcriptase (Promega, Madison, WI) with a random hexamer as a primer in a 20-µl reaction. For PCR amplification, 3 µl of cDNA reaction product was used with 10 µM CDK6-specific primers that span an intron to eliminate amplification from genomic DNA: CDK6 417, 5'-CCGAGTAGTGCATCGCGATCTAA-3'; CDK6 823, 5'-CTTTGCCTAGTTCATCGATATC-3'.

As a loading control, 18 S RNA was amplified from the same samples. To adjust the amplification of 18 S cDNA, primer pairs for 18 S RNA were mixed with a 1:9 ratio of 18 S competimer pairs (Ambion, Austin, TX), which are primers with a modified 3' end to block extension. For PCR amplification, 50 µl of PCRs (1× Taq polymerase buffer Mg2+-free, 0.2 mM dNTPs, 0.25 µl of Taq polymerase (Life Technologies, Inc.), 1.5 mM MgCl2, 0.2 µM each primer) were amplified for 30 cycles (95 °C, 30 s/55 °C, 30 s/68 °C, 30 s). The PCRs were precipitated with 1/10th volume of 3 M sodium acetate and 2.5× volume of 100% ethanol, rinsed with 70% ethanol, resuspended in 1× STR loading dye (32% formamide, 3.3 mM NaOH, 0.05% bromphenol blue, 0.05% xylene cyanol), and separated on a 6% acrylamide gel with 7 M urea. To visualize the DNA, the acrylamide gel was fixed in 10% acetic acid, stained with 0.1% silver nitrate, 0.06% formaldehyde, and developed with 3% sodium carbonate, 0.06% formaldehyde. NIH image was used to quantify the silver-stained bands and adjust for loading as determined by 18 S cDNA levels.

Flow Cytometric Analysis of DNA Content-- MCF-7 cells were plated at 40,000 cells/well of a 6-well tissue culture dish and treated for 48 h in complete media. I3C was added to a final concentration of 200 µM, tryptophol to 200 µM, tamoxifen to 1 µM, and actinomycin D to 1 µg/ml. The media were changed every 24 h. Following treatment, cells were hypotonically lysed in 1 ml of DNA staining solution (0.5 mg/ml propidium iodide, 0.1% sodium citrate, 0.05% Triton X-100). Cell debris was removed by filtration through 60-µm nylon mesh (Sefar America Inc., Kansas City, MO). Nuclear-emitted fluorescence with wavelengths of >585 nm was measured with a Coulter Elite instrument. Ten thousand nuclei were analyzed from each sample at a rate of 300-500 nuclei/s. The percentages of cells within the G1, S, and G2/M phases of the cell cycle were determined by analysis with the Multicycle software MPLUS (Phoenix Flow Systems) in the Cancer Research Laboratory Microchemical Facility of the University of California, Berkeley.

LipofectAMINE Transfection-- For reporter assays in MCF-7 cells, transfections were performed by mixing 3 µl of LipofectAMINE (Life Sciences, St. Petersburg, FL) and 1 µg of reporter plasmid. LipofectAMINE reactions were quenched after 5 h by addition of 2× media. After 24 h cells were incubated in complete media. For basal activity assays, cells were collected 48 h after transfection. For construction of stable cell lines, cells were also transfected with 0.1 µg of pCMV-Neo and selected with 800 µg/ml geneticin (G418 sulfate, Life Technologies, Inc.), in complete media for 3 weeks, followed by propagation in 400 µg/ml geneticin in complete media. After selection, stable pools of transfected cells or clonally derived cell lines were plated at 40,000 cells/well of a 6-well dish and incubated in complete media with or without 1 µl/ml Me2SO, 200 µM indole-3-carbinol, 200 µM tryptophol, 1 µM tamoxifen, or 1 µg/ml actinomycin D depending on the experimental protocol.

Luciferase Assay-- For luciferase assays, cells were harvested by washing twice in phosphate-buffered saline and lysed in 100-200 µl of 1× Promega reporter lysis buffer. 20 µl of MCF-7 cell lysate was added to 12 × 75 mm cuvettes (Analytical Luminescence Laboratory, San Diego, CA) and subsequently loaded into a luminometer (Monolight 2010, Analytical Luminescence Laboratory). 100 µl of luciferase substrate buffer (20 mM Tricine, 1.07 mM (MgCO3)4Mg(OH)25H2O, 2.67 mM MgSO4, 0.1 mM EDTA, 33.3 mM DTT, 270 µM coenzyme A, 470 µM D-luciferin sodium salt, 530 µM ATP disodium salt, pH 7.8) was injected automatically into each sample, and luminescence was measured in relative light units. The luciferase specific activity was expressed as an average of relative light units produced per µg of protein present in corresponding cell lysates as measured by the Bradford assay (Bio-Rad).

Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assay-- Preparation of nuclear extracts from MCF-7 cells was based on a method described previously (37). Briefly, cells were hypotonically lysed in Buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.5 mM PMSF), allowed to swell for 30 min, gently homogenized in a Dounce homogenizer, and centrifuged at 550 rpm to isolate nuclei. Nuclei were homogenized with a micro-pestle and then incubated in Buffer C (20 mM HEPES, pH 7.9, 25% glycerol, 420 mM NaCl, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF) for 30 min on a rocking platform to strip DNA-binding proteins. After centrifugation at 14,000 rpm, the resulting supernatant was dialyzed against Buffer D (20 mM HEPES, pH 7.9, 20% glycerol, 1.5 mM MgCl2, 100 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF) for 2.5 h and then frozen at -70 °C. The protein content in the nuclear extracts was normalized by the Bradford procedure (38). All gel shift oligonucleotides were ordered from MWG-Biotech (Ebersberg, Germany) and high pressure liquid chromatography-purified as follows: for probe -680/-638, wt Ets/wt Sp1, 5'-GTCTCCCTGCACGTCCGGGCCTCAGGAGGCGGGGAGGGTAGC-3'; for competitors, wt Ets/wt Sp1, 5'-GTCTCCCTGCACGTCCGGGCCTCAGGAGGCGGGGAGGGTAGC-3'; wt Ets/mut Sp1, 5'- GTCTCCCTGCACGTCCGGGCCTCAGGAGCCATGGAGGGTAGC-3'; mut Ets/wt Sp1, 5'- GTCTCCCTGCCATGGCGGGCCTCAGGAGGCGGGGAGGGTAGC-3'; mut Ets/mut Sp1, 5'- GTCTCCCTGCCATGGCGGGCCTCAGGAGCCATGGAGGGTAGC-3'; consensus Ets/consensus Sp1, 5'-GTCTCCCTGCACTTCCGGGCCTCAGGGGGCGGGGAGGGTAGC-3'; consensus Sp1, 5'-ATTCGATCGGGGCGGGGCGAGC-3'.

Radiolabeling of the 5' ends of oligonucleotide probes was carried out in the presence of equal amounts (10 pmol) of sense and antisense strands, [gamma -32P]ATP (6000 Ci/mmol and T4 polynucleotide kinase (Roche Molecular Biochemicals)) at 37 °C for 40 min followed by annealing of the labeled strands by adding 0.1 M NaCl to the reaction mix, heating 10 min at 70 °C, and gradually cooling to room temperature. The free unincorporated nucleotides and single-stranded DNA were separated from the end-labeled double-stranded DNA by native polyacrylamide gel electrophoresis on an 8% gel with 0.5× TBE (50 mM Tris, pH 8, 45 mM borate, 0.5 mM EDTA). The labeled double-stranded oligonucleotide was excised and eluted in 400 µM TE (10 mM Tris, 1 mM EDTA) and 40 µl of 3 M sodium acetate, pH 5.0, overnight. DNA-binding reactions (18 µl) contained nuclear extract proteins (~20 µg), 15,000 cpm DNA probe, 2 µg of poly(dI-dC), and 6 µl of 3× binding buffer (60 mM HEPES, pH 7.9, 7.5 mM MgCl2, 36% glycerol, 3 mM DTT, 3 mM EDTA, 180 mM KCl), and were incubated for 20 min on ice before being resolved on a 4% native polyacrylamide gel (19:1 acrylamide/bisacrylamide) in 1× TGE (25 mM Tris, pH 7.9, 190 mM glycine, 1.3 mM EDTA) at 4 °C, 170 V. For competition experiments, a 100-fold excess of the indicated unlabeled double-stranded oligonucleotide was added prior to the addition of radiolabeled DNA probe and incubated for 20 min on ice. For the supershift assay indicated, binding reactions contained 1 µg of anti-Sp1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) incubated on ice for 20 min before addition of radiolabeled probe. Protein-DNA complexes in the dried gels were visualized by autoradiography using Kodak BioMax film (PerkinElmer Life Sciences).

Western Blot Analysis-- After the indicated treatments, cells were harvested in RIPA buffer (150 mM NaCl, 0.5% deoxycholate, 0.1% Nonidet P-40, 0.1% SDS, 50 mM Tris) containing protease and phosphatase inhibitors (50 µg/ml PMSF, 10 µg/ml aprotinin, 5 µg/ml leupeptin, 0.1 µg/ml NaF, 1 mM dithiothreitol (DTT), 0.1 mM sodium orthovanadate and 0.1 mM beta -glycerophosphate). Equal amounts of total cellular protein were mixed with loading buffer (25% glycerol, 0.075% SDS, 1.25 ml of 14.4 M 2-mercaptoethanol, 10% bromphenol blue, 3.13% stacking gel buffer) and fractionated by electrophoresis on 10% polyacrylamide, 0.1% SDS resolving gels. Rainbow marker (Amersham Pharmacia Biotech) was used as the molecular weight standard. Proteins were electrically transferred to nitrocellulose membranes (Micron Separations, Inc., Westborough, MA) and blocked overnight at 4 °C with 5% non-fat dry milk in 1× western wash buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20). Blots were subsequently incubated with antibodies against CDK6, which were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The antibody concentration was 1 µg/ml in western wash buffer. Immunoreactive proteins were detected after 1-3 h of incubation at room temperature with horseradish peroxidase-conjugated secondary antibodies. Goat anti-rabbit at secondary antibodies were used (Bio-Rad) after being diluted to 3 × 10-4 in western wash buffer with 1% non-fat dry milk. Blots were treated with ECL reagents (PerkinElmer Life Sciences), and the proteins were detected by autoradiography. Equal protein loading was ascertained by Ponceau S staining of blotted membranes.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

I3C Repression of CDK6 Transcript Levels Is Not Due to Changes in RNA Stability-- We have reported previously that the I3C-induced G1 cell cycle arrest of MCF-7 human breast cancer cells is accompanied by a selective decrease in mRNA and protein level for the G1 cyclin-dependent kinase CDK6, with no effect on CDK2 or CDK4 expression (10). A Western blot of MCF-7 cells treated with or without 200 µM I3C for up to 72 h shows that the inhibition of CDK6 protein levels is observed by 24 h of indole treatment, with a near-maximal effect by 48 h of treatment (Fig. 1, panel A). To begin to determine the cellular processes responsible for the I3C down-regulation of CDK6 expression within the first 24 h of indole treatment, such as transcriptional versus post-transcriptional mechanisms, the potential effect of I3C on CDK6 mRNA stability was characterized in MCF-7 cells. Cells were treated with or without 200 µM I3C in the presence or absence of 1 µg/ml actinomycin D, a transcriptional inhibitor, for various times, and the level of CDK6 mRNA was quantified by relative reverse transcriptase-PCR. This dose of actinomycin D inhibits greater than 90% of [3H]uridine incorporation.2 If I3C treatment decreases the stability of CDK6 mRNA, CDK6 transcripts isolated from cells treated with both actinomycin D and I3C should have a shorter half-life than those from cells treated with actinomycin D alone. Silver staining of the PCR-amplified cDNA revealed that treatment with I3C or actinomycin D alone or with both reagents caused a rapid loss of CDK6 transcripts compared with cells treated with the Me2SO vehicle control (Fig. 1, panel B). The level of silver-stained bands was quantified and adjusted for loading as determined by 18 S cDNA levels. As also illustrated in Fig. 1 (panel C), there is a transient and somewhat variable increase in the levels of CDK6 transcripts upon treatment of cells with fresh media. In untreated cells, CDK6 transcripts return to basal levels within 3-5 h; therefore, the level of CDK6 sequences at 5 h was set to 1.0 and used to normalize decay curves for CDK6 transcripts under each experimental condition. The observed half-life of CDK6 mRNA was ~8 h in cells treated with I3C or actinomycin D alone, or exposed to both inhibitors (Fig. 1, panel C). Thus, I3C treatment does not further reduce CDK6 transcript levels in cells where the transcription has been inhibited, suggesting the I3C response is not due to alterations in the half-life of CDK6 transcripts. Although formally possible that actinomycin D treatment blocks synthesis of a protein needed for degradation of CDK6 transcripts, treatment with I3C appears not to alter CDK6 mRNA stability in MCF-7 cells. It is important to point out that the use of silver staining to detect the reverse transcriptase-PCR products of CDK6 transcripts is a semi-quantitative method that cannot distinguish relatively minor alterations in transcript levels. These results suggest that any changes in CDK6 transcript stability play a minimal, if any, role in the I3C inhibition of CDK6 gene expression and indicate that the I3C response is mediated by a transcriptional mechanism. To test this possibility directly, we cloned the CDK6 gene promoter to determine if I3C regulates CDK6 promoter activity.


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Fig. 1.   Effects of indole-3-carbinol on CDK6 transcript decay rate in MCF-7 cells. Panel A, MCF-7 cells were treated with or without 200 µM I3C for the indicated times. Total cell extracts were electrophoretically fractionated in SDS-polyacrylamide gels, and Western blots were probed with primary antibodies to CDK6. Panel B, MCF-7 cells were treated with or without 200 µM I3C in the presence or in the absence of 1 µg/ml actinomycin D (Act D) for 0, 3, 5, 8, 15, or 24 h. The isolated RNA was converted to cDNA, amplified by PCR, and electrophoretically fractionated, and polyacrylamide gels were silver-stained to reveal PCR products as described under "Experimental Procedures." As a loading control, 18 S cDNA, a constitutively expressed transcript, was amplified from the same samples. Panel C, the relative level of CDK6 transcripts shown in panel A were quantitated as described under "Experimental Procedures." The reported values in panel B were calculated, after correcting for loading using the 18 S cDNA, as the percentage of the level of CDK6 cDNA in the Me2SO vehicle control at each time point. Open circles indicate the vehicle control treated cells; × indicates cells treated with 200 µM I3C in the presence of 1 µg/ml actinomycin D; dark boxes indicate cells treated with 1 µg/ml actinomycin D; and dark circles indicate cells treated with 200 µM I3C.

Cloning, Identification, and Characterization of the cdk6 Promoter and Analysis of cdk6 Gene Structure-- The cloning and characterization of the CDK6 gene promoter has not been reported previously. By using a BLAST search of the EST data base, the CDK6 gene was identified as EST SWSS3105, which had been mapped to chromosome 7q21. This region of the human genome has been cloned into BAC and sequenced by the human genome sequencing project. The BAC containing exon 1 of CDK6 and 6000 bp of the CDK6 5'-flanking region was identified by searching a data base of BACs known to span the 7q21 region and obtained from Genome Systems. No differences were detected between the exonic genomic sequence reported by the Human Genome Project and the previously reported cDNA sequence for CDK6 (39). CDK6 is composed of 7 exons spanning ~200 kb (Fig. 2). The signature PLSTIRE helix, which contributes a critical glutamic acid residue to the active site and is partially responsible for cyclin and p16 binding, is found within exon I. Exon III contains the T-loop, which is regulated by phosphorylation by the cell cycle kinase CAK. Exons III and IV contain the two segments of the kinase domain, and Exon VII contains the stop codon (40). The six introns ranged in size from 2785 to 58,259 bp. The deduced genomic organization the CDK6 locus is consistent with a previous report comparing the mouse and human CDK6 loci (41).


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Fig. 2.   Schematic representation of the human CDK6 gene locus. Organization of the CDK6 locus was determined by aligning the CDK6 complete cDNA sequence (GenBankTM X66365) with the genomic sequence that has been established for the chromosome 7q21 region. cDNA segments aligning with the CDK6 genomic sequence are represented by dark rectangles and intervening intronic sequences by solid lines, and the length of each segment in base pairs is indicated. The 2462-bp region of the CDK6 promoter used in this study is represented by a solid line to the left of the first exon.

The CDK6 promoter was isolated using a PCR-based strategy and sub-cloned into PGL2-basic luciferase expression vectors. The sequence of the CDK6 promoter from -920 bp through +119 bp of the first exon is shown in Fig. 3. The sequence of the entire promoter is listed in GenBankTM (accession number AF332591). To determine if the 5'-upstream region of the CDK6 gene could function as a promoter, deletion fragments ranging from -2464 to -196 bp cloned into the promoter-less pGL2-basic luciferase reporter vector. After transient transfection into MCF-7 cells, basal luciferase activity of constructs -450 bp and larger averaged 100 times higher than activity of the pGL2-basic vector alone (Fig. 3). The -196-bp fragment averaged only 2-fold greater activity than the pGL2-basic vector. Similar results were obtained with transient transfection into MDA-MB-231 breast cancer cells.2 The -638-bp fragment consistently exhibited the greatest amount of luciferase activity, perhaps indicating the loss of a repressive element.


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Fig. 3.   Promoter region of the human CDK6 gene and basal activity of promoter fragments in MCF-7 cells. Panel A, the sequence of the CDK6 promoter and part of the first exon is shown from -920 to +119. The * shows the RNA start site, and the last 3 base pairs represent the ATG protein start site. The sequence of the CDK6 promoter out to -2464 is listed in GenBankTM with accession number AF332591. Panel B, the diagram shows a schematic representation of the CDK6 gene promoter, with potential transcription factor-binding sites indicated by dark ovals. The 5' end of each promoter fragment used in this study is indicated. Panel C, reporter gene activity of 5' deletion constructs was analyzed by luciferase assay in transiently transfected MCF-7 cells. The reported values are representative of four independent experiments of triplicate samples, and the error bars indicate the standard deviation. RLU, relative light units.

Consensus transcription factor-binding sites within the cloned -2464 CDK6 promoter were identified using the TRANSFAC data base searched with MatInspector (42), and the TFMATRIX transcription factor-binding site profile data base was searched with TFSEARCH (Fig. 3). Sites for several cell cycle-related transcription factors are found in the CDK6 promoter, including NFkappa B, E2F, and Ets family transcription factors. The CDK6 gene promoter does not contain a TATA box, similar to other TATA-less cell cycle genes such as cdc2 (43), CDK2 (34), and cyclin A1 (44). The Sp1 transcription factor, which binds to GC-rich regions, is known to guide initiation in some TATA-less promoters (45, 46). There are segments of the CDK6 promoter with GC-rich regions as long as 20 bp in length, and there is an apparent CpG island, which in other promoters facilitates the unfolding of chromatin structure for access to transcription factors (47).

MCF-7 human breast cancer cells produce extremely low levels of CDK6 mRNA that have made mapping the 5' end of the RNA transcript technically challenging. Repeated efforts to map the RNA start site by RNase protection and primer extension assays have been unsuccessful. We have isolated various clones from the 5'-rapid amplification of cDNA ends procedure, none of which extend beyond the published 5' end of the CDK6 cDNA (39), possibly because of the high GC content in this region of the 5'-untranslated region (>90%). For this study, the most 5' nucleotide of the published CDK6 cDNA sequence is designated +1, and by this convention, all deletion constructs have their 3' termini at +24.

I3C Inhibition of cdk6 Promoter Activity and Specificity of the Response-- To determine whether I3C can down-regulate CDK6 promoter activity, MCF-7 cells were stably transfected with the -920-bp CDK6-luciferase reporter plasmid. The transfected cells were treated with or without I3C for 48 h in complete media and then assayed for luciferase activity and for cell cycle distribution by flow cytometry of propidium iodide-stained nuclei. As illustrated in Fig. 4, under conditions in which I3C caused a significant increase in cells with a G1 phase DNA content, CDK6 promoter activity was strongly down-regulated. Treatment with tryptophol, which is closely related to I3C, containing an ethanol group instead of a methanol group in the 3-carbon position, neither altered CDK6 promoter activity nor induced a G1 arrest of MCF-7 cells (Fig. 4, Tryp versus I3C). This negative result with tryptophol suggests there is a cellular selectivity in responsiveness to extracellular indole compounds. Multiple MCF-7 cell lines were derived from both single cell subclones and pools of cells transfected with the -920 CDK6-luciferase reporter plasmid. In each of these cell lines, I3C inhibited CDK6 promoter activity to approximately the same extent2 indicating that the observed promoter regulation was not due to clonal variation of the transfected cells. Treatment of transfected cells with the actinomycin D transcriptional inhibitor under conditions that reduce by >95% total incorporation of [3H]uridine down-regulated the CDK6 promoter only slightly more strongly than I3C, suggesting that I3C causes a near-maximal inhibition of CDK6 promoter activity. In later experiments shown in this study, the luciferase activity observed in the presence of actinomycin D is used as the assay background.


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Fig. 4.   Indole-3-carbinol treatment specifically down-regulates CDK6 promoter activity. Panel A, MCF-7 cells stably transfected with the -920-bp CDK6-luciferase reporter plasmid were treated with 200 µM I3C, 200 µM tryptophol (Tryp), 1 µM tamoxifen (Tam), 1 µg/ml actinomycin D (Act D), or with the vehicle control for 48 h and assayed for luciferase activity as described under "Experimental Procedures." The relative light units per µg of protein are representative of three independent experiments of triplicate samples, and the error bars indicate the standard deviation. Panel B, MCF-7 cells were treated with 200 µM I3C, 200 µM tryptophol, 1 µM tamoxifen or with the vehicle control for 48 h. Cells were then stained with propidium iodide, and nuclei were analyzed for DNA content by flow cytometry with a Coulter Elite Laser. A total of 10,000 nuclei was analyzed from each sample. The percentages of cells within the G1 phase of the cell cycle were determined as described under "Experimental Procedures." The reported values are an average of at least three independent experiments, and the error bars indicate standard deviation.

MCF-7 cells are estrogen-responsive, and these cells can be growth-inhibited by the anti-estrogen tamoxifen in complete medium (28). This phenotype allowed us to test if the regulation of CDK6 promoter activity is specific to I3C treatment or a general consequence of the growth-arrested state of the cells. In parallel with the above-mentioned experiments, the -920-bp CDK6-luciferase transfected cells were also treated for 48 h with 1 µM tamoxifen. Although tamoxifen arrested MCF-7 cells in the G1 phase of the cell cycle to approximately the same extent as I3C, no changes were observed in CDK6 promoter activity (Fig. 4). Consistent with this result, we have shown previously that tamoxifen had no effect on CDK6 protein or mRNA levels (28). In addition, treatment of MCF-7 cells with the synthetic glucocorticoid, dexamethasone, caused a substantial increase in G1 phase arrested cells but did not alter CDK6 promoter activity.2 Taken together, these results demonstrate that the I3C inhibition of CDK6 promoter activity is a specific effect of this indole and not a consequence of the growth-arrested state of the cells.

Deletion Analysis of the cdk6 Promoter Defines a 167-bp I3C-responsive Region-- To functionally define the cis-acting region of the CDK6 promoter that confers responsiveness to I3C, serial 5' deletions of the promoter were constructed, cloned into luciferase reporter plasmids, and stably transfected into MCF-7 cells. After 3 weeks of selection, CDK6 promoter activity and the cell cycle arrest were examined in stable pools of transfected cells treated with or without I3C for 48 h. As shown in Fig. 5, activity of deletion constructs containing fragments of the CDK6 promoter ranging from -2462 to -805 bp are all significantly reduced by I3C treatment (Student's t test p <=  0.05 for the -2462, -920, and -805-bp fragment), whereas -638- and -450-bp CDK6 promoter fragments failed to respond to I3C. I3C treatment of each of the corresponding stable pools of cells also resulted in growth arrest in the G1 phase of the cell cycle and down-regulation of CDK6 protein expression.2 This series of deletion constructs uncovered an I3C-responsive region that can be localized within the 167-bp sequence from -805 to -638 bp of the CDK6 promoter.


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Fig. 5.   Deletion analysis of the CDK6 promoter defines a 167-bp indole-3-carbinol-responsive region. MCF-7 breast cancer cells were stably transfected with a series of CDK6-luciferase reporter plasmids that contain the indicated 5' deletions of the CDK6 promoter. Cells were treated with or without 200 µM I3C for 48 h, and the luciferase specific activity was determined as the luciferase (Luci) activity produced per µg of protein present in the corresponding cell lysates relative to the vehicle control (see "Experimental Procedures"). The reported values are representative of at least four independent experiments of triplicate samples, and the error bars indicate the standard deviation.

Mutation of the Ets or Sp1 DNA-binding Sites within the I3C-responsive Region Ablates I3C Responsiveness of the cdk6 Promoter-- Sequence analysis of the I3C-responsive region by the TFMATRIX transcription factor-binding site profile data base revealed the presence of putative transcription factor-binding sites for NFkappa B, Ets, and Sp1 families of transcription factors (all in the antiparallel direction). To determine which of these cis-acting elements potentially plays a role in the I3C down-regulation of CDK6 promoter activity, each of three individual sites was mutated to a NcoI restriction enzyme site (CCATGG) in the context of the -920-bp CDK6-luciferase reporter plasmid. The corresponding mutant and the wild type -920 CDK6-luciferase reporter plasmids were stably transfected into MCF-7 cells and assayed for I3C-responsive promoter activity. As demonstrated in Fig. 6, I3C treatment significantly reduced luciferase activity in cells expressing the wild type or the NFkappa B mutant promoters but had virtually no effect on constructs containing the Sp1 and Ets mutants. These results suggest that intact Sp1-like and Ets-like elements in the CDK6 promoter are necessary at a functional level for the I3C signaling pathway to down-regulate CDK6 gene expression. I3C failed to inhibit reporter plasmid activity of MCF-7 cells transfected with a Sp1-chloramphenicol acetyltransferase reporter construct encoding three consensus Sp1 DNA-binding sites,2 suggesting that I3C responsiveness requires the context of the CDK6 promoter. The Sp1 and Ets sites are separated by only 5 bp of intervening sequence (Fig. 5), and it is tempting to consider that they may be working in concert to mediate I3C responsiveness of the CDK6 promoter.


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Fig. 6.   Site-directed mutagenesis of NFkappa B, Ets, and Sp1 DNA-binding sites within the indole-3-carbinol-responsive region of the CDK6 promoter. MCF-7 breast cancer cells were stably transfected a series of CDK6-luciferase reporter plasmids that contain the indicated transcription factor binding site mutations. In these -920-bp CDK6 promoter-luciferase reporter plasmids, NFkappa B, Ets, and Sp1-like DNA-binding sites were mutated by PCR to NcoI restriction endonuclease sites. Cells were treated with or without 200 µM I3C for 48 h, and the luciferase (Luci) specific activity was determined as the luciferase activity produced per µg of protein present in the corresponding cell lysates relative to the vehicle control (see "Experimental Procedures"). The reported values are an average of at least three independent experiments of triplicate samples, and the error bars indicate standard deviation.

I3C Treatment Reduces Binding of Nuclear Proteins to the I3C-regulated Region of the cdk6 Promoter-- A gel mobility shift assay was used to characterize biochemically nuclear protein-DNA interactions within the I3C-regulated region of the CDK6 promoter. Nuclear extracts were prepared from MCF-7 cells that had been treated with or without with 200 µM I3C for 48 h and incubated with a 32P-labeled double-stranded oligonucleotide that corresponds to a 42-bp segment of the I3C-responsive region (-680 to -638 bp). The extracted nuclear proteins bind to this CDK6 promoter fragment to form a series of retarded bands, the largest of which is significantly reduced in intensity by I3C treatment (indicated by an arrow on Fig. 7). Interaction of proteins with this CDK6 promoter fragment increased over a range of extracted nuclear protein from 5 to 20 µg in the binding reaction. At each concentration of protein, the gel-shifted protein-DNA complexes formed using nuclear extracts from I3C-treated cells was reduced by 5-10-fold compared with the extracts isolated from growing vehicle control-treated cells.


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Fig. 7.   Indole-3-carbinol treatment reduces the intensity of a large gel-shifted DNA complex. Nuclear extracts were prepared from MCF-7 breast cancer cells that had been treated with either 200 µM I3C or the vehicle control for 48 h. Extracts (5, 10, or 20 µg) were incubated on ice for 20 min with a radiolabeled -680/-638-bp fragment of the CDK6 promoter that contains the composite Ets and Sp1-like DNA-binding elements. The protein-DNA complexes were resolved on low ionic strength native 6% polyacrylamide gels.

Identification of Sp1 as an I3C-regulated Trans-acting Factor That Targets the I3C-responsive Region of the cdk6 Promoter-- A competitive gel shift analysis was used to initially investigate whether the Sp1 and/or Ets transcription factor DNA sites contribute to the formation of DNA-protein complexes within the I3C-responsive region of the CDK6 promoter. Combinations of the Sp1 and Ets DNA-binding sites were mutated within a 42-bp section of the I3C regulated region (-680 to -638 bp). The extracted nuclear proteins were incubated with 100-fold excess of unlabeled wild type or mutant promoter fragments prior to addition of the radiolabeled wild type promoter fragment. As shown in Fig. 8, the wild type and Ets only mutant promoter fragments efficiently competed with the radiolabeled wild type promoter fragment for formation of the major gel-shifted complexes. In contrast, oligonucleotides containing a mutant Sp1-binding site alone, or in combination with an Ets site mutation, failed to compete with the wild type promoter fragment. In a complementary approach, incubation with an oligonucleotide containing consensus the Sp1 DNA-binding site alone, or along with a consensus Ets-binding site, also strongly competed with the radiolabeled wild type CDK6 promoter fragment for formation of the protein-DNA complexes (Fig. 8, last two lanes). Taken together, these results indicate that the Sp1 site in the I3C-responsive region of the CDK6 promoter is required for formation of a predominant protein-DNA complex.


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Fig. 8.   Competitive gel shift analysis of the -680/-638-bp region of the CDK6 promoter. Nuclear extracts were prepared from MCF-7 breast cancer cells that had been treated with either 200 µM I3C or the vehicle control for 48 h. Extracts (20 µg) were preincubated with a 100× excess of unlabeled oligonucleotide. Oligonucleotides derived from the -680/-638-bp region of the promoter were synthesized with wild type or mutant versions of the Ets-like and/or Sp1-like-binding sites and are described under "Experimental Procedures." Following a 20-min incubation, radiolabeled wild type -680/-638-bp fragment was added to each sample and allowed to incubate further for 20 min on ice. The protein-DNA complexes were resolved on low ionic strength native 6% polyacrylamide gels.

Antibody supershift assays were used to demonstrate the presence of Sp1 protein in the I3C-responsive protein-DNA complex. Nuclear proteins were extracted from cells treated with or without I3C for 48 h and incubated with a radiolabeled oligonucleotide corresponding to the wild type -680 to -638-bp region of the CDK6 promoter in the presence or absence of anti-Sp1 antibodies. As shown in Fig. 9, inclusion of the anti-Sp1 antibodies induced a supershift (see *) of the highest molecular weight protein-DNA complex (see arrow). Furthermore, nuclear protein extracts isolated from I3C treated cells displayed a significant down-regulation of the anti-Sp1 antibody-supershifted protein-DNA complex. These results demonstrate the presence of Sp1 in the I3C-regulated protein-DNA complex that forms on this CDK6 promoter fragment.


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Fig. 9.   Addition of anti-Sp1 antibody causes a supershift in the indole-3-carbinol-regulated gel shift complex. Nuclear extracts were prepared from MCF-7 breast cancer cells that had been treated with either 200 µM I3C or the vehicle control for 48 h. Protein extracts (20 µg) were incubated with either 1 µg of polyclonal anti-Sp1 antibody (Sp1 Ab+ lanes) or 1 µg of rabbit IgG (Sp1 Ab- lanes) for 20 min on ice. Following antibody incubation, reactions were incubated on ice for 20 min with the radiolabeled -680/-638-bp fragment of the CDK6 promoter. The protein-DNA complexes were resolved on low ionic strength native 6% polyacrylamide gels.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Elucidation of the signal transduction pathways and identification of final gene targets by which cell cytostatic agents mediate their effects can identify candidate gene products that may be exploited for the further development of therapeutic strategies. I3C is a promising chemotherapeutic agent for treatment of breast cancers because of its potent growth inhibitory properties (4, 5, 10, 28) and in vitro repression of invasion and migration of cultured breast cancer cell lines (48). We have documented previously that I3C induced a G1 cell cycle arrest of MCF-7 breast cancer cells that is accompanied by the inhibition of CDK6 transcript and protein levels and total cellular CDK6 kinase activity (10). These results suggest that I3C could potentially control the emergence and proliferation of breast cancer cells through its cell cycle effects, although relatively little is known about the molecular details of this pathway.

Our current work has now established a direct link between I3C signaling and the control of CDK6 expression in MCF-7 breast cancer cells. I3C down-regulates CDK6 transcription by targeting Sp1 transcription factor binding to a composite Ets-Sp1 DNA site within the I3C-regulated region of the CDK6 promoter. Transfection of wild type and mutant CDK6 promoter fragments in luciferase reporter plasmids demonstrated that the Sp1 and Ets DNA sites were both necessary for the I3C inhibition of CDK6 promoter activity. Electrophoretic gel mobility shift assays revealed that the interaction of the Sp1 transcription factor with the Sp1 DNA element of the composite Ets-Sp1 DNA site in the CDK6 promoter is responsible for forming an I3C-inhibited protein-DNA complex. Based on our results, we propose that the transcriptional regulation of CDK6 is a critical early step in the G1 block in cell cycle progression induced by I3C in human breast cancer cells. This study not only describes the initial cloning and identification of the human CDK6 promoter but is also the first example of the regulation of CDK6 transcription through specific cis-acting promoter elements.

Sp1 is a ubiquitously expressed transcription factor (49) that binds to GC-rich regions found in many genes. There are many examples of ubiquitous transcription factors interacting with tissue-specific transcription factors to selectively control gene expression. Sp1 and its family members can play an important role in regulating tissue-specific gene expression (44, 50). Depending on the promoter context, Sp1 can function as a regulator of basal transcription by binding near the transcriptional start site (45, 46) or it can act as a transcriptional activator by binding to upstream enhancer elements (51). Sp1 interacts with a variety of other transcriptional regulators, including members of the nuclear receptor superfamily. For example, Sp1 interacts with the aryl hydrocarbon receptor, which has several xenobiotic ligands including dioxin (52). In addition, a multiprotein complex containing the estrogen receptor, Sp1, and aryl hydrocarbon receptor has been implicated in regulation of cathepsin D gene expression in MCF-7 cells (53). Estrogen induces expression of several genes, including E2F1 (54), IGFBP4 (55), adenosine deaminase (56) and c-FOS (57) via ER-Sp1 interactions (58) at GC-rich elements. Sp1 also interacts with the androgen receptor to regulate the response of p21 to androgens (59). These studies suggest that Sp1 interactions with other transcriptional regulators may control the specificity of the I3C mediated down-regulation of CDK6 promoter activity. In this regard, activity of the CDK6 promoter fragment containing the composite Ets-Sp1 site is down-regulated by I3C, although transfection of a reporter plasmid driven by three consensus Sp1 sites was unresponsive to this indole.2

The close proximity (5 bp) of the Ets-like site to the I3C-responsive Sp1 site in the CDK6 promoter suggests that a member of the Ets-like family may be a candidate Sp1-interacting transcription factor. Members of the Ets family of transcription factors are critical nuclear integrators of signaling pathways controlling cell proliferation, differentiation, and oncogenic transformation (60, 61). Specificity of gene regulation by Ets family transcription factors is achieved through subsets of cell type-specific family members, by selective interactions with partner proteins, and modulation of activity through phosphorylation events (62). Consistent with our studies, Ets and Sp1 have been shown to interact in a variety of systems. For example, Ets- and Sp1-related factors cooperate to regulate the tissue inhibitor of metalloproteinases-1 metalloproteinase promoter in fibroblasts (63), the CD18 (beta 2 leukocyte integrin) promoter in leukocytes (64), and the protein kinase ck2alpha gene promoter in HeLa cells (65). We propose that I3C regulates the CDK6 promoter through a combination of transcription factors involving protein-protein and protein-DNA interactions between members of the Ets and Sp1 transcription factor families, possibly interacting with other transcription factors within the -638-bp CDK6 promoter fragment. Our observation that the Ets-like binding site is important for the I3C down-regulation of CDK6 promoter activity, but does not contribute to DNA binding, indicates that binding to the Ets-like DNA element may potentially be stabilized by protein/protein interactions outside of the region covered by the gel shift probe and that these interactions are important for the complete responsiveness of the CDK6 promoter to I3C. Consistent with this possibility, a 300-bp fragment of the CDK6 promoter containing the I3C-regulated region is not sufficient to confer I3C responsiveness to two different heterologous minimal promoters, the 56-bp c-FOS or 75-bp Sgk promoters.2

Cell type- and stimulus-specific regulation of transcription involves the combinatorial interactions of DNA-bound transcription factors and non-DNA-binding transcriptional regulators (66). The histone acetylase-deacetylase protein complexes are an important class of interacting factors that do not directly bind to DNA (67). Interestingly, the Sp1 protein is known to recruit the DRIP-TRAP coactivator complex (68), which plays an essential role in transcriptional activation by nuclear receptors and may also regulate other types of transcriptional responses (69). Many transcriptional coactivators have histone acetylase activity (70), and their characterization has made a mechanistic connection between histone acetylation and gene expression. Conversely, histone deacetylation plays a role in the repression of transcription by allowing a more compact chromatin structure and less access of transcription factors to the DNA (67). Given our observation that the I3C suppression of CDK6 promoter activity occurs only in integrated reporter plasmids, and not in transiently transfected cells, it is likely that chromatin structure plays a role in the I3C responsiveness of the CDK6 promoter.

Our study provides the basis for further studies of transcriptional regulation by I3C and for a more comprehensive understanding of the I3C-induced cell cycle arrest of human breast cancer cells. Further characterization of the I3C-regulated region in the CDK6 promoter, including protein-protein interactions involving Sp1, will allow us to identify critical transcription factors that are downstream effectors of I3C, and elucidate aspects of the mechanism by which I3C growth arrests human breast cancer cells. In addition, because of the relatively high concentration of I3C needed to inhibit CDK6 promoter activity and induce a G1 cell cycle arrest, we are also attempting to identify synthetic and natural derivatives of I3C that act at significantly lower doses than I3C. This information could ultimately facilitate further clinical trials of dietary indoles leading to the development of indole-based therapeutics that control breast cancer cell proliferation.

    ACKNOWLEDGEMENTS

We thank Douglas P. Finkbeiner, Hanh H. Garcia, and Anita C. Maiyar for their critical evaluation of this manuscript and helpful experimental suggestions. We also thank Karin E. Hansen for help in preparing the final figures that accompany this study, as well as other members of both the Firestone and Bjeldanes laboratories for their helpful comments throughout the duration of this work. We thank Minnie Wu, Tim Labadie, and Cindy Huynh for their technical assistance.

    FOOTNOTES

* This work was supported in part by by Department of Defense Army Breast Cancer Research Program Grant DAMD17-96-1-6149, by the University of California Breast Cancer Research Program Grant 31B-0110 (to G. L. F.), and by United States Public Health Service Grant CA69056-05 from the NCI, National Institutes of Health (to L. F. B).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by Department of Defense Army Breast Cancer Research Program Predoctoral Grant BC971062.

|| To whom correspondence and reprint requests should be addressed: Dept. of Molecular and Cell Biology, 591 LSA, University of California, Berkeley, CA 94720-3200. Tel.: 510-642-8319; Fax: 510-643-6791; E-mail: glfire@uclink4.berkeley.edu.

Published, JBC Papers in Press, April 10, 2001, DOI 10.1074/jbc.M010539200

2 E. J. Cram and G. L. Firestone, unpublished data.

    ABBREVIATIONS

The abbreviations used are: I3C, indole-3-carbinol; CDK, cyclin-dependent kinase; Me2SO, dimethyl sulfoxide; Sp1, promoter specificity factor 1; NFkappa B, nuclear factor kappa B; BLAST, basic local alignment sequence tool; EST, expressed sequence tag; BAC, bacterial artificial chromosome; bp, base pair; PCR, polymerase chain reaction; DTT, dithiothreitol; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PMSF, phenylmethylsulfonyl fluoride.

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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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