From the 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
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ABSTRACT |
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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 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.
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. [ 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:
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
Primers used for construction of mutant 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
Radiolabeling of the 5' ends of oligonucleotide probes was carried out
in the presence of equal amounts (10 pmol) of sense and antisense
strands, [ 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 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.
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).
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
Consensus transcription factor-binding sites within the cloned
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
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 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 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 NF 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 ( 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 (
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 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 ( 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.
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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.
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'.
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.
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'; NF
B mut F,
5'-TCCTGGCCATGGCCACCCACATTCTGC-3'; and NF
B mut R,
5'-GGTGGCCATGGCCAGGACCCTGTAACC-3'.
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'.
-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).
-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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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.
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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.
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.
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 NF
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).
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.
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.
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.
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 NF
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
NF 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, NF
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.
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.
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.
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
2 leukocyte
integrin) promoter in leukocytes (64), and the protein kinase
ck2
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
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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.
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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.
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ABBREVIATIONS |
---|
The abbreviations used are:
I3C, indole-3-carbinol;
CDK, cyclin-dependent kinase;
Me2SO, dimethyl sulfoxide;
Sp1, promoter specificity factor
1;
NFB, nuclear factor
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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REFERENCES |
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