Estrogen Up-Regulation of p53 Gene Expression in MCF-7 Breast Cancer Cells Is Mediated by Calmodulin Kinase IV-Dependent Activation of a Nuclear Factor
B/CCAAT-Binding Transcription Factor-1 Complex
Chunhua Qin,
Thu Nguyen,
Jessica Stewart,
Ismael Samudio,
Robert Burghardt and
Stephen Safe
Department of Veterinary Physiology & Pharmacology (C.Q., T.N., J.S., I.S., R.S.) and Department of Veterinary Anatomy and Public Health (R.B.), Texas A&M University, College Station, Texas 77843-4466
Address all correspondence and requests for reprints to: Stephen H. Safe, Department of Veterinary Physiology & Pharmacology, Texas A&M University, 4466 TAMU, College Station, Texas 77843-4466. E-mail: ssafe{at}cvm.tamu.edu.
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ABSTRACT
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This study investigates the mechanism of hormonal regulation of p53 gene expression in MCF-7 human breast cancer cells. 17ß-Estradiol (E2) induced a 2-fold increase in p53 mRNA levels and a 2- to 3-fold increase in p53 protein. Analysis of the p53 gene promoter has identified a minimal E2-responsive region at -106 to -40, and mutation/deletion analysis of the promoter showed that motifs that bind CCAAT-binding transcription factor-1 (CTF-1) and nuclear factor
B (NF
B) proteins are required for hormone responsiveness. The p65 subunit of NF
B was identified in both nuclear and cytosolic fractions of untreated MCF-7 cells; however, formation of the nuclear NF
B complex was E2 independent. Hormonal activation of constructs containing p53 promoter inserts (-106 to -40) and the GAL4-p65 fusion proteins was inhibited by the intracellular Ca2+ ion chelator EGTA-AM and Ca2+/calmodulin-dependent protein kinase (CaMK) inhibitor KN-93. Constitutively active CaMKIV but not CaMKI activated p65, and treatment of MCF-7 cells with E2 induced phosphorylation of CaMKIV but not CaMKI. The results indicate that hormonal activation of p53 though nongenomic pathways was CaMKIV-dependent and involved cooperative p65-CTF-1 interactions.
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INTRODUCTION
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THE p53 TUMOR suppressor gene plays an important role in regulating cell proliferation and the cell cycle and activates growth inhibitory pathways and apoptosis (reviewed in Refs. 1, 2, 3, 4). Activation of p53 by UV damage or other agents/signals results in p53-mediated transcription or up-regulation of genes such as the cyclin-dependent kinase inhibitor p21, bax and FAS (apoptotic genes), and GADD45, an enzyme involved in DNA repair. Mechanisms associated with p53-mediated responses are complex and dependent on cell/tissue context. For example, p53 directly modulates gene expression as a nuclear transcription factor that binds as a homodimer to p53 response elements in regulatory regions of several genes including bax, p21 and GADD45 (5, 6, 7). p53 represses transcriptional activation by direct protein-protein interactions with TATA binding protein and other proteins involved in DNA synthesis. p53 also inhibits transactivation by binding DNA elements to block DNA replication (8, 9, 10, 11, 12). p53 mutations have been extensively characterized and correlated with increased incidence of multiple cancers including breast cancer. p53 mutations have been also associated with poor prognosis for breast cancer patients and many p53 mutations are missense mutations in the DNA binding domain (reviewed in Refs. 2, 13, 14, 15, 16, 17).
p53 is expressed in estrogen receptor
(ER
)- positive T47D breast cancer cells and levels are significantly decreased in cells maintained in charcoal-stripped serum or serum-free media (18, 19, 20). 17ß-Estradiol (E2) has minimal effects on p53 levels in cells grown in serum-containing media, whereas antiestrogens such ICI 164,384, tamoxifen or 4'-hydroxytamoxifen decrease p53 protein. However, in T47D cells grown in media containing charcoal-stripped serum, E2 significantly increased levels of p53 protein (18). It was also reported that E2 induced chloramphenicol acetyltransferase (CAT) activity in T47D or MCF-7 breast cancer cells transfected with the PICAT construct that contains a 2.4-kb insert from the p53 gene promoter linked to a CAT reporter gene (19). In the present study, we have investigated the mechanism of E2-mediated transcriptional activation of p53 in MCF-7 human breast cancer cells. Treatment with E2 increased p53 mRNA (
2-fold) and protein (2- to 3-fold) levels and luciferase (luc) reporter gene activity in MCF-7 cells transfected with p531 (containing the -1800 to +12 region of the p53 gene promoter). Subsequent deletion and mutational analysis of the p53 gene promoter has identified a minimal 67-bp region (-106 to -40) that is required for hormonal activation of p53-derived constructs and the key cis-acting elements include CTF-1 and nuclear factor
B (NF
B) binding sites where CTF-1 acts as a DNA-bound cofactor for NF
B through direct interactions with p65. E2 activates p53 through nongenomic pathways and studies with kinase inhibitors show that p65 is activated in breast cancer cells through E2-dependent activation of calmodulin kinase IV (CaMKIV), and this is consistent with a recent report showing that CaMKIV also activates p65 in CV-1 and HeLa cells (21).
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RESULTS
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1) Transcriptional Activation of p53 and Deletion Analysis of the p53 Gene Promoter
Previous studies have demonstrated that p53 protein levels are increased in ER-positive breast cancer cells after treatment with E2 (18, 19, 20), and the results in Fig. 1A
also show that a time-dependent increase in p53 mRNA levels was observed after treatment with E2 and a maximum 2-fold increase was noted after 20 h. Ten- and 100-nM E2 alone significantly induced (1.7- to 2.2-fold) luc activity in MCF-7 cells transfected with p53-1, which contains a p53 gene promoter insert from -1800 to +12 (Fig. 1B
). Moreover, after cotransfection with ER
, there was an enhanced (7.8-fold) induction of reporter gene activity after treatment with E2. Enhanced gene expression after cotransfection with ER
has previously been reported using other E2-responsive constructs (22, 23, 24, 25, 26), and ER
was cotransfected in subsequent experiments in this study.

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Figure 1. Transcriptional Activation of p53 mRNA by E2
A, Northern blot analysis of p53 mRNA levels in MCF-7 cells. Cells were seeded in 5% CSS DMEM/F12 media for 24 h, and in serum-free DMEM/F12 media for another 48 h, and treated with DMSO (vehicle) or E2 in fresh serum-free media as indicated. The mRNA levels were determined as described under Materials and Methods. E2 significantly (*, P < 0.05) induced p53 mRNA levels after 20 h treatment. Results for all transfection studies are expressed as mean ± SD for three replicate determinations for each treatment group. B, Effects of E2 treatment on p53 gene promoter-luc reporter activity in MCF-7 cells. Cells were transiently transfected with 2.5 µg of p53 gene promoter-luc reporter construct (p53-1) or empty vector pGL2, 1 µg of ß-gal expression vector, with (indicated as "+") or without (indicated as "-") 1 µg of ER expression plasmid for 20 h, and then treated for 2448 h with DMSO or 10 nM E2. Luciferase activity was determined as described in Materials and Methods, expressed as units (U) and normalized to ß-gal activity. The empty vector pGL2 was included in all transfections as a control. Results are expressed as mean ± SD, and significant (*, P < 0.05) induction was observed for E2 treatment alone. Cotransfection with ER further boosted induction to 7.8-fold. All comparisons are made within the same experiment.
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Deletion analysis of the p53 gene promoter demonstrated that basal activity and E2 responsiveness were differentially modulated by deletions of the 5'-regulatory region from -1800 (p53-1) to -44 (Fig. 2A
). Deletion of the -1800 to -1199 region increased basal activity (p53-1/p53-2) and further deletion to -592 (p53-3) resulted in a 4-fold decrease in basal activity in MCF-7 cells. Basal activity increased 2-fold in cells transfected with p53-4, suggesting inhibitory elements within the -1800 to -1199 and -592 to -312 region of the p53 gene promoter. Marked loss of luc activity was observed in cells transfected with p53-7, p53-8 and p53-9 containing the -66 to +12, -44 to +12, and -106 to -67 p53 gene promoter inserts. E2 responsiveness was observed in cells transfected with p53-1 to p53-6 (3- to 7.8-fold) indicating that the -106 to +12 region of the promoter was the minimal sequence required for hormone activation. Moreover, a 3'-deletion to give p53-9 (-106 to -67) resulted in loss of E2 responsiveness.

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Figure 2. Deletion and Mutation Analysis of p53 Constructs in MCF-7 Cells Identified a Minimal Sequence (from -106 to -40) that Is Essential for E2 Induction
A, 5' Sequential deletion analysis indicated that the fragment (from -106 to +12) is required for E2 responsiveness. Cells were transiently transfected with 2.5 µg of p53 gene promoter-luc reporter constructs (p53-1 to -9), 1 µg of ß-gal expression plasmid, and 1 µg of ER expression plasmid for 20 h, and then treated for 2448 h with DMSO or 10 nM E2. Luciferase activity was determined as described in Materials and Methods and normalized to ß-gal activity. B, Further analysis of the p53 short promoter (from -106 to +12) identified a minimal E2-responsive sequence (from -106 to -40). MCF-7 cells were transiently transfected with 2.5 µg of p53-6 to -14 constructs, and some mutants as indicated by "X", and cotransfected with 1 µg of ER and 1 µg of ß-gal expression plasmids as described above. Cells were treated with DMSO or 10 nM E2 for 2448 h, and significant induction (P < 0.05) by E2 is indicated by an asterisk.
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2) Identification of p53 Promoter Elements Required for E2 Responsiveness
Deletion analysis of the p53 gene promoter indicated that the -106 to +12 region (p53-6) is required for E2 responsiveness, and this sequence contains putative binding sites for CTF-1/YY1, nuclear factor-Y (NF-Y), NF
B, E2F, Sp1-like proteins, and an E-box (Fig. 2B
). Mutation of the E2F binding site (p53-6 m) or deletion of the -30 to +12 region of the promoter (p53-10) did not result in loss of E2 responsiveness, suggesting that the E2F site is not required for E2 action. Deletion of the 5' CTF-1/YY1 (p53-11 and p53-12) and mutation or deletion of the NF
B (p53-10 m2 and p53-14) binding sites resulted in loss of E2-induced transactivation, whereas mutation or deletion of the overlapping GC-rich/E-box motifs (p53-10 m1 and p53-13) did not affect this response. These results suggest that hormone-induced transcriptional activation of constructs derived from the p53 gene promoter require the -106 to -40 region containing intact CTF-1/YY1 and NF
B binding sites.
3) Protein Interactions with the p53 Gene Promoter
Protein interactions with the E2-responsive region of the p53 gene promoter were determined using nuclear extracts from MCF-7 cells and [32P]-labeled oligonucleotides from -106 to -30 (Fig. 3
). Preliminary gel mobility shift studies indicated that ER
did not directly bind this region of the p53 promoter. The upstream fragment (from -106 to -67) has been reported to bind both CTF-1 and YY1 (27). Results in Fig. 3B
show that nuclear extracts bind [32P]-106/-67 to form one major retarded band (lane 1) and intensity of this band is decreased only after competition with 50-fold excess of unlabeled -106/-67 and consensus CTF-1 oligonucleotides (lanes 2 and 4), but not by consensus oligonucleotides that bind NF-Y, CP2, C/EBP, NF
B, Sp1 (lanes 3, 57, and 9) or an estrogen-responsive element (ERE) (lane 8). ER
does not interact with this region (-106 to -67) of the promoter and oligonucleotide competition experiments suggest that CTF-1 binds this sequence. In a separate experiment, it was shown that [32P]-labeled YY1 probe formed a band with MCF-7 cell nuclear extracts; however, the unlabeled YY1 oligonucleotide did not competitively decrease binding to [32P]-106/-67 (data not shown).

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Figure 3. CTF-1, NF-Y, NF B, Sp1, and Sp3 Proteins Bind to the Sequence from -106 to -30
Binding of nuclear extracts to oligonucleotides and antibody supershifts were determined by EMSA, as described in Materials and Methods. A, The p53-10 insert is shown and putative CTF-1, NF-Y, NF B, and GC-rich sites are indicated (CTF-1, NF-Y, and GC-rich sites are underlined; NF B is italicized). The p106/67 and p66/30 oligonucleotides used in gel shift assays contain the -106 to -67 and -66 to -30 regions of the p53 gene promoter, respectively. B, Competition studies with [32P]-p106/67. Nuclear extract from E2-treated MCF-7 cells bound [32P]-p106/67 and consensus CTF-1 oligonucleotide competitively decreased the band. C, Competition studies with [32P]-p66/30. Using MCF-7 cell extracts in a binding buffer containing ZnCl2, Sp1/Sp3, NF B, and NF-Y oligonucleotides competitively decreased bands labeled A, B/C, and D, respectively. D, Competition studies with [32P]-p66/30. The gel shift assay was carried out in buffer which does not contain ZnCl2 and does not form a Sp1/Sp3 DNA complex. Bands B/C and D were competitively decreased after competition with NF B, and NF-Y oligonucleotides, respectively. E, Antibody supershift experiments. NF-Y, Sp1, and Sp3 antibodies supershifted their corresponding retarded bands ("A" and "D") in Sp1-binding buffer (as in Fig. 3C ). F, NF B p65 and p50 supershifted their related retarded bands (B and C) in the buffer without ZnCl2. Preimmune IgG was included as a negative control.
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Binding of MCF-7 cell nuclear extracts to the downstream [32P]-66/-30 region of the promoter gave a more complex pattern of four retarded bands (lane 1), and competition with 100-fold unlabeled -66/-30 (lane 2), consensus NF
B (lane 4), and Sp1 (GC-rich) (lane 5) oligonucleotides decreased intensity of the retarded bands (Fig. 3C
). The higher molecular weight band A was preferentially decreased by coincubation with the consensus Sp1 oligonucleotide, whereas intensities of the lower molecular weight bands B and C were preferentially decreased by the consensus NF
B oligonucleotide. The lowest molecular weight band D was affected (decreased intensity) only after competition with consensus NF-Y oligonucleotide (lane 3), whereas competition with an ERE did not affect intensities of bands AD. Using slightly altered binding conditions that favor NF-Y but not Sp1 binding (band A) (Fig. 3D
), only bands B, C, and D were formed, and results of unlabeled oligonucleotide competition experiments were similar to those observed in Fig. 3C
. The failure of an ERE to decrease band intensities indicates that ER also does not bind directly to the -66 to -30 region of the p53 gene promoter.
Confirmation of specific protein interactions with [32P]-66/-30 were also determined in antibody supershift experiments (Fig. 3E
) using the two incubation conditions described in Fig. 3
, C and D. In this experiment where bands A, B, C, and D were observed (lane 1, Fig. 3E
), supershift experiments with Sp1 and Sp3 antibodies (lanes 3 and 4) gave at least two supershifted bands, and NF-YA antibodies also gave a supershifted complex (lane 5). Using incubation conditions that eliminated Sp1-DNA complex formation (Fig. 3F
) (lane 1), antibodies to p65 and p50 proteins supershifted bands B and C, respectively (lanes 3 and 4). Results from antibody supershift experiments confirm oligonucleotide competition studies showing that NF-Y, Sp1, Sp3, p65, and p50 bind to the overlapping NF-Y/NF
B/GC-rich sites in the -66 to -30 region of the p53 gene promoter.
4) Physical and Functional Interactions of NF
B and CTF-1
Interactions of CTF-1 and p65 with the p53 gene promoter were also investigated using a chromatin immunoprecipitation assay in which cells were treated with formaldehyde to form DNA-protein cross-links. After sonication, and immunoprecipitation by p65 or CTF-1 antibodies, PCR was used to determine binding of p65 and CTF-1 protein to the -196 to -6 region of the p53 gene promoter (Fig. 4A
). The results indicated that both p65 and CTF-1 were bound to the promoter in untreated cells and in cells treated with E2 for 15 min; the slight decrease in transcript levels observed after 15 min may be due to several factors including decreased antigen recognition due to recruitment of other promoter binding proteins after treatment with E2. cAMP response element binding protein (CREB) antibodies were used as a negative control for the p53 promoter immunoprecipitation and transcripts were not detected. In a separate experiment, we used a modified chromatin immunoprecipitation (ChIP) assay method to investigate interactions of CaMKIV, ER
, and Sp1 interactions with the proximal region of the p53 gene promoter (Fig. 4B
). This modified assay used an increased number of cells to isolate immunoprecipitable chromatin complexes, and PCR products were separated by agarose gel electrophoresis and detected by ethidium bromide staining as described (28). CaMKIV was cross-linked to the p53 gene promoter, and both ER
and Sp1 also bound to this region of the promoter. Detection of Sp1 binding is consistent with the GC-rich motif in the promoter. In addition, we have previously detected ER
associated with GC-rich promoters in the presence or absence of E2 (Refs. 29, 30 , and our unpublished results), and this is consistent with the reported ligand-independent direct interactions between ER
and Sp1 proteins (31, 32). In this study, ER
only weakly interacted with the p53 gene promoter in untreated cells; however, enhanced interactions were observed after treatment with E2 for up to 120 min. As an additional control experiment, we also observed ER
interactions with the cathepsin D gene promoter (-294 to -54) after 0, 15, and 30 min (Fig. 4C
), whereas Sp1 protein was bound after 30 min (positive control). This results is consistent with the role of ER
and Sp1 proteins in activation of cathepsin D (25), and previous studies (33) have also reported the ER
antibodies immunoprecipitate this region of the cathepsin D gene promoter. In contrast, ER
and Sp1 antibodies did not immunoprecipitate a region of exon 2 of the cathepsin D gene (negative control) (Fig. 4D
).

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Figure 4. ChIP Assay for Proteins Interacting with the p53 and Cathepsin D Gene Promoters
A, p53 gene promoter. PCR primers targeted to the-196 to-6 region of the p53 gene promoter were used to determine p54, CTF-1 and CREB immunoprecipitable complexes as described in Materials and Methods. [32P]Deoxy-CTP incorporation was used to detect PCR products illustrated in A, C, and D. Using a modified PCR approach (B) in which bands were detected by ethidium bromide staining (28 ), cross-linked complexes immunoprecipitated by ER , CaMKIV, and Sp1 antibodies were determined as described in the Materials and Methods. As a control, the ChIP assay was also carried out using the E2-responsive region of the cathepsin D promoter (-294 to -54) (C) and a region in exon 2 of cathepsin D (D).
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Because both NF
B and CTF-1 motifs on the p53 gene promoter are required for estrogen action (Figs. 1
and 2
), we examined the interactions of these proteins in immunoprecipitation (Fig. 5A
) and a mammalian two-hybrid assay (Fig. 5B
). In coimmunoprecipitation assays (Fig. 5A
), [35S]p65 protein (lane 1) was immunoprecipitated by p65 antibody (lane 3), whereas this same antibody did not immunoprecipitate [35S]CTF-1 (lane 5). However, after incubation of [35S]CTF-1 with p65 protein, p65 antibody immunoprecipitated [35S]CTF-1 (lane 4), demonstrating physical interactions between p65 and CTF-1 proteins. Whole cell lysates from E2-treated MCF-7 cells were immunoprecipitated with CTF-1 antibody (lane 8) or with IgG as a control (lane 7). Lane 8 was whole cell lysate alone (Fig. 5A
). All samples were analyzed by Western blot analysis with p65 antibody, which showed positive immunostaining in lanes 7 and 8. Cellular interactions between p65 and CTF-1 were investigated in a mammalian two-hybrid system (Fig. 5B
) in MCF-7 cells. p65 or a C-terminal region of p65 (p65c1) were fused to the DNA binding domain (DBD) of the yeast GAL4 protein in the pM vector. CTF-1 was fused to the acidic VP16 protein in the pV16 vector, and the cells were also transfected with a construct containing 5 tandem GAL4 response elements (pGAL4-luc). Transfection with the GAL4 fusion proteins containing p65 (pM-p65) or p65c1 (pM-p65c1) increased luc activity by 256- and 483-fold, respectively. Cotransfection with pV-CTF-1 decreased activities of the fusion proteins by 6571%, thus indicating that non-DNA-bound CTF-1 squelched pM-p65 and pM-p65c1-mediated activities. In contrast, pV-CTF-1 did not affect the activity of pM3VP16, indicating that the interaction between CTF-1 and p65 was specific. Thus, CTF-1 appears to act as a cofactor for p65 in the context of the p53 gene promoter.

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Figure 5. Physical and Functional Interactions of CTF-1 and NF B p65 Proteins
A, Coimmunoprecipitation of p65 and CTF-1. Coimmunoprecipitation was performed as described in Materials and Methods, and lanes 1 and 2 show [35S]-labeled in vitro translated NF B and CTF-1 proteins, respectively. NF B p65 antibody precipitated [35S]-labeled NF B (lane 3), and also precipitated [35S]-labeled CTF-1 through interaction of CTF-1 with unlabeled NF B (lane 4). B, Functional interactions of NF B p65 and CTF-1 in a mammalian two-hybrid system. The NF B p65 or p65 C-terminal 267 amino acids (p65c1) were fused with GAL4 DBD of pM vector (CLONTECH Laboratories, Inc.). CTF-1 was fused with VP16 of pV vector (CLONTECH Laboratories, Inc.); 2.5 µg of 5X Gal4 reporter (pGAL4-luc) was cotransfected with 0.5 µg of pM/pM fusion and 0.5 µg pV/pV fusion proteins and 1 µg of ß-gal. Transfection with pM-p65 and pM-p65c1 resulted in 256- and 483-fold increase in luc activity compared with pM/pV control, respectively (compare C to A, or E to A, P < 0.05).
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5) Activation of p5310 (-106 to -30) by E2 and Affects on Subcellular Distribution of I
B and NF
B
The E2 responsive -106 to -30 region of the p53 gene promoter does not bind ER, suggesting that hormonal activation may be due to ER
interactions with other DNA-bound transcription factors such as Sp1 or AP1 (29, 30, 31, 32, 34, 35, 36, 37) or through nongenomic induction of kinase pathways (38, 39, 40, 41, 42, 43, 44). Transcriptional activation of p5310 by E2 in MCF-7 cells was not affected by cotransfection with dominant negative (dn) NF-Y and Sp1 expression plasmids (Fig. 6A
), and the latter result is consistent with the E2 responsiveness of p5310 m1, which is mutated in the GC-rich Sp1 binding site. These results suggest that, although ER
and Sp1 can interact with this region of the p53 gene promoter (Fig. 4B
), ER
/Sp1 interaction with the GC-rich site is not required for hormone-induced transactivation. In contrast, activation of p5310 by E2 was significantly inhibited after cotransfection with the I
B super repressor (I
B-SR), which cannot be phosphorylated and degraded (45) and after cotransfection with precursor-derived inhibitor (pdI), which contains the C-terminal region of the p105 precursor of p50 protein component of the NF
B heterodimer. Purified pdI can dissociate preformed complexes of bacterial p50 bound to DNA and also inhibit purified NF
B (46). In addition, the proteasome inhibitor MG132 also blocked transcriptional activation of p5310 by E2. The results obtained for MG132, pdI and I
B-SR are consistent with an important role for the NF
B site in transcriptional activation of the p53 gene promoter by E2, and this was supported by the hormone nonresponsiveness of p5310 m2 (Fig. 2
) containing an NF
B site mutation. The importance of the NF
B pathway in hormonal activation of p53 was further confirmed in studies showing that E2 induced a 2- to 3-fold increase in p53 protein levels after treatment for 2 or 6 h, and the induction response was blocked in cells transfected with I
B-SR (Fig. 6B
).

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Figure 6. Role of I B and NF B DNA Binding in Hormonal Activation of p53 by E2
A, I B degradation and binding of NF B to DNA are required for E2 induction. Transient transfection was performed as described in Materials and Methods using 2.5 µg of p5310, 1 µg of ER, 1 µg of ß-gal and 0.25 µg of either I B-SR/pdI/dnNF-Y/dnSp1 constructs. A 10-µM concentration of MG-132 was used to pretreat MCF-7 cells for 2 h before treatment with E2. I B-SR, pdI and MG-132 completely blocked E2-induced luc activity, whereas dnNF-Y and dnSp1 did not affect the induction response using up to 2 µg of dnNF-Y and dnSp1 (data not shown). B, Effects of E2 and I B-SR on p53 protein levels. Whole cell lysates from MCF-7 cells treated with DMSO (C) or E2 in the presence or absence of cotransfected I BSR were analyzed for p53 protein by Western blot analysis as described in the Materials and Methods. E2 induced p53 protein levels by 2- to 3-fold (2 and 6 h), and this response was blocked with I B-SR. C, I B degradation and NF B nuclear translocation in MCF-7 cells treated with E2 or IL-1ß. Western blot analysis was performed as described in Materials and Methods. D, Effects of E2 and IL-1ß on NF B-DNA binding. Treatment with IL-1ß for 15 and 30 min significantly increased NF B binding to DNA (lanes 3 and 4), whereas E2 did not affect NFkB-DNA binding (lanes 8 and 9). Petreatment with CHX inhibited I B resnythesis, and thus reversed decreased NF B binding to DNA (compare lane 6 to lane 5). Consensus NF-Y and NF B oligonucleotide competition and p65 antibody supershifts (lanes 1012) were employed as controls. E, Increased NF B binding to DNA is not sufficient for induction of p53 by E2. Transient transfection was performed as described in Materials and Methods using 2.5 µg of p5310, 1 µg of ER, 1 µg of ß-gal. Group 1 was treated with DMSO or E2, group 2 was pretreated with 1 ng/ml IL-1ß for 2 h before treatment with DMSO or E2, and group 3 was treated with DMSO or IL-1ß. F, Immunostaining of p65 in MCF-7 cells. Cells were stained with p65 antibodies as described in Materials and Methods. In cells treated with DMSO, approximately 40% of the cells exhibited nuclear p65 staining (top) and this was only slightly enhanced (60%) (bottom) after treatment with E2.
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Because hormonal activation of p53 is dependent on nuclear NF
B, it is possible that E2 either induces formation of nuclear NF
B or directly activates NF
B (p65/p50) through nongenomic pathways that involve induction of kinase activities (38, 39, 40, 41, 42). The responsiveness of MCF-7 cells to other activators of NF
B was investigated using IL-1ß, which caused a significant decrease in cytosolic I
B protein after treatment for 15 or 30 min (but not 60 min). Recovery of protein levels after 60 min was blocked by cycloheximide (CHX) (Fig. 6C
, lane 9). In contrast, E2 had no effect on levels of cytosolic I
B or nuclear p65 protein (Fig. 6C
, lanes 6 and 7), whereas IL-1ß increased nuclear p65 protein levels after treatment for 15, 30, or 60 min (Fig. 6C
, lanes 35). NF
B binding to the -60 to -30 region of the p53 gene promoter was analyzed by gel mobility shift assay (Fig. 6D
), and the results showed that the IL-1ß alone (lanes 25) or in combination with cycloheximide (lane 6) increased binding, whereas cycloheximide alone (lane 7) or E2 alone (lanes 8 and 9) did not induce NF
B binding. Binding specificity was demonstrated by competition with unlabeled consensus NF
B oligonucleotide and antibody supershifts (lanes 11 and 12); in addition, unlabeled NF-Y oligonucleotide decreased intensity of the NF-Y-DNA complex band. Although IL-1ß significantly increased the formation of the DNA-protein complex, treatment with this cytokine did not induce luc activity in MCF-7 cells transfected with p5313 and did not affect the E2-induced response (Fig. 6E
). These data suggest that treatment of MCF-7 cells with E2 did not markedly affect nuclear NF
B levels of DNA binding.
Initial studies in MCF-7 cells showed that I
B protein was detected in nuclear and cytosolic extracts and was unaffected by treatment with E2 (data not shown). Application of fluorescence imaging techniques using p65 antibodies showed significant nuclear staining in untreated cells, and this was only slightly enhanced after treatment with E2 (Fig. 6F
). These results are consistent with the results of transactivation, Western blot and gel mobility shift assays indicating that E2 does not activate p53 by inducing nuclear accumulation of NF
B.
6) E2 Directly Activates NF
B through the C-Terminal Activation Domain of p65
The direct activation of p65 and CTF-1 by E2 through nongenomic pathways were investigated using GAL4 fusion proteins and a pGAL4-luc construct in MCF-7 cells. The results in Fig. 7A
show that E2 directly activates p65 C-terminal transactivation domain, but not CTF-1 in one-hybrid system indicating that p65 is the major downstream target of E2. p65c2 contains the C-terminal 30 amino acids from p65 fused to the GAL4-DBD; this construct was cotransfected with pGAL4-luc and ER, and treatment with E2 induced 3.8-fold increase in luc activity. This response was not blocked by cotransfection with pdI indicating that E2 directly activated the p65 C-terminal transactivation domain that does not interact with pdI. E2 did not induce luc activity through pM-CTF-1 or pM-CTF-1c (a fusion of GAL4-DBD with CTF-1 C-terminal 100 amino acids), and CTF-1 or CTF-1c showed less transactivation potential than NF
B under these conditions in MCF-7 cells.

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Figure 7. Role of CaMKIV in Activation of p53-10/pM-p65c2 in MCF-7 Cells
A, pdI does not block activation of pM-p65c2. MCF-7 cells were transfected with pGAL4-luc, pM, pM-p65c2, pM-p65c2 plus pdI, pM-CTF-1 or pM-CTF-1c, treated with DMSO ( ) or 10 nM E2 ( ), and luc activity was determined as described in Materials and Methods. Luciferase activity was significantly (P < 0.05) activated in the DMSO group (*) and induced by E2 (**). Effects of kinase inhibitors and EGTA on hormonal activation of pM-p65c2 (B) and p53-10 (C). Cells were transfected with pM-p65c2 or p53-10 and treated with E2, DMSO alone or in the presence of different kinase inhibitors or EGTA, and luc activity was determined as described in Materials and Methods. Significant (P < 0.05) induction by E2 is indicated by an asterisk. Results are presented as means ± SD for at least three replicate experiments for each treatment group. D, Activation of pM-p65c2 by constitutively active CaMKI and CaMKIV. MCF-7 cells were transfected with pM-p65c2 alone and in combination with CaMKIc or CaMKIVc (constitutively active) (0.1 or 0.5 µg) and luc activity determined as described in Materials and Methods. Significant (P < 0.05) induction is indicated (*), and results are express as mean ± SD for three replicate determinations for each treatment group. E, Phosphorylation of CaMKIV. MCF-7 cells were pretreated with [32P]orthophosphate for 3 h and then treated with DMSO (C) or 10 nM E2 for 2, 30 or 240 min; whole cell lysates were obtained, immunoprecipitated (IP) with CaMKIV antibody, and analyzed by SDS-PAGE/Western blot (WB) analysis and phosphoimaging as described in Materials and Methods. Increased phosphorylation of CaMKIV after treatment with E2 for 30 min was observed in duplicate experiments. In parallel studies using CaMKI antibodies, phosphorylation of CaMKI was not observed.
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Kinase-dependent activation of p53 by E2 was further investigated in MCF-7 cells transfected with pM-p65c2/pGAL4-luc or p5310 and treated with dimethylsulfoxide (DMSO) or 10 nM E2 and the following kinase inhibitors: 50 µM PD98059 (MAPKK), 3 µM SB203580 (p38 kinase), lavendustin and 10 µM PP2 (protein tyrosine kinase), 800 µM SQ 22536 (adenylate cyclase), 15 µM H89 and 500 nM KT5720 (protein kinase A), 100 nM wortmannin (phosphatidylinositol-3-kinase), 1 µM rapamycin (p70S6K), and 3 µM Gö6850 (protein kinase C). None of these kinase inhibitors blocked hormonal activation of pM-p65c2 or p5310 and similar results were obtained for LY294002 and damnacanthal (data not shown). The results illustrated in Fig. 7
, B and C, show that 3 µM KN-93 does not block hormonal activation of pM-65c2 or p5310, whereas 30 µM KN-93 and the intracellular calcium ion chelator EGTA-AM blocked induction in cells transfected with the same constructs. A recent report indicated that 30 µM KN-93 inhibited Ca2+/CaM-dependent protein kinase-mediated activation of p65 in CV-1 and HeLa cells. Moreover, both constitutively active CaMKI and CaMKIV (but not CaMKII) also activated p65, and CaMKIV directly associated with p65 in vivo and in vitro (21). In contrast, our results show that constitutively active CaMKIVc but not CaMKIc activated pM-65c2 in MCF-7 cells (Fig. 7D
). Furthermore, E2 also significantly induced phosphorylation of CaMKIV after treatment for 30 min (Fig. 7E
). However, in parallel studies, increased phosphorylation of CaMKI was not observed even though CaMKI was detected by Western blot analysis (data not shown). These results show that hormonal activation of p65/NF
B is dependent on E2-induced release of intracellular Ca2+ and subsequent phosphorylation of CaMKIV. These observations are consistent with previous reports showing that E2 activated intracellular Ca2+ in MCF-7 cells (40) and CaMKIV activated p65 in CV-1 and HeLa cells (21).
 |
DISCUSSION
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Several studies report that E2 induces p53 mRNA/protein levels in breast cancer cells, and reporter gene activity was also induced in cells transfected with a construct containing a 2.4-kb insert from the p53 gene promoter (18, 19, 20). Deletion analysis of a series of constructs containing p53 gene promoter inserts demonstrates that maximal constitutive reporter gene activity in MCF-7 cells was observed with p53-2 containing the -1199 to +12 region of the promoter. There was a 7585% decrease in basal activity observed in cells transfected with p53-6 (-106 to +12) or p53-10 (-106 to -30), and this was increased in p53-13 (-106 to -40) suggesting that deletion of the GC-rich and upstream stimulatory factor sites enhanced basal activity (Fig. 2
). Deletion of the CTF-1 site did not decrease basal activity of p53-12 (-80 to -30), whereas interaction of CTF-1 (or YY1) with this site was important for basal activity of the p53 promoter in HeLa cells (27). Previous studies have reported that ER
/Sp1 and NFYA interactions with GC-rich and CCAAT sites, respectively, were necessary for induction of E2F1 in MCF-7 cells (34); however, these interactions were not required for hormone activation of p53-10 (Fig. 2
). Although gel mobility shift analysis (Fig. 3
) and chromatin immunoprecipitation assays (Fig. 4
) confirmed that multiple proteins in MCF-7 cell nuclear extracts bound the -106 to -30 region of p53 gene promoter, results of transactivation studies (Fig. 2
) suggest that the NF
B and CTF-1 sites are required for E2 responsiveness and protein binding to the NF
B site is also critical for basal activity (Fig. 2
).
The CTF-1 binding site in the minimal promoter of p53 (-106 to -40) is conserved in human, rat, and mouse p53 promoters. Occupancy of this site varies in a tissue-specific manner; it is bound primarily by YY1 in nuclear extracts of rat testis and spleen and by CTF-1 in extracts of liver and prostate, and this may facilitate tissue-specific control of p53 gene expression. Both YY1 and CTF-1 function as activators when bound to this site in HeLa cells (27). However, in this study, only CTF-1 bound this element (Fig. 3
) and in a chromatin immunoprecipitation assay with CTF-1 antibodies (Fig. 4
), there was direct evidence for CTF-1 (and NF
B) binding to the proximal region of the p53 gene promoter. Previous studies have shown that ER
can activate a chimeric protein containing the proline-rich C-terminal domain of CTF-1 fused to the GAL4 DBD in transient transfection studies in HeLa cells; however, these results were obtained using a promoter-reporter construct containing a ERE insert (47). In MCF-7 cells, we did not observe hormonal activation of CTF-1 using pGAL4-luc and either pM-CTF-1 (full coding sequence) or pM-CTF-1c (fusion with C-terminal 100 amino acids). However, when fused to pVP16, the CTF-1 fusion protein significantly squelched activity of pM-p65 (Fig. 5
), indicating that CTF-1 interacts in vivo with p65. Immunoprecipitation data using whole cell lysates and in vitro expressed p65 and CTF-1 also confirmed physical interaction of these proteins (Fig. 5A
), suggesting that DNA-bound CTF-1 acts as cofactor of NF
B for activation of p53. In contrast, the NMDA receptor agonist quinolinic acid also induced p53 in neuronal cells through NF
B; however, this response was dependent on increased nuclear uptake of p65 and did not require CTF-1 as a cofactor (48).
A major pathway for activation of NF
B signaling involves phosphorylation of I
B followed by dissociation of the NF
B-I
B complex and rapid proteasome-dependent degradation of I
B (reviewed in Refs. 49 49 and 50). This is also accompanied by the rapid nuclear translocation of NF
B and formation of a nuclear NF
B complex. This was also observed after treatment of MCF-7 cells with IL-1ß (Fig. 6
, B and C) and previously described for this cytokine in other cell lines (49, 50). Activation of p53 constructs and p53 protein by E2 requires dissociation of I
B and proteasome-dependent degradation (Fig. 6
, A and B), but increased binding of nuclear NF
B complex to DNA was not observed after treatment with E2. In contrast, IL-1ß enhanced NF
B binding to the p53 promoter, but did not induce luc activity or enhance E2-induced luc activity in cells transfected with p5310. These results indicate that E2 does not enhance formation of nuclear NF
B. Immunohistochemistry studies (Fig. 6F
) indicate that p65 is nuclear in approximately 40% of untreated MCF-7 cells, and this percentage is only slightly increased (approximately 60%) after treatment with E2. This suggests that E2 may directly activate NF
B in MCF-7 cells.
Other studies have demonstrated that treatment-induced dissociation of cytosolic I
B from NF
B is not sufficient for activation through NF
B site; moreover, degradation of I
B and NF
B-DNA binding are not always correlated with activation of target genes (51, 52, 53, 54). For example, Bergmann et al. (51) showed that activation of an NF
B-dependent reporter gene by TNF and IL-1ß in A549 human alveolar cells is blocked by phospholipase C and protein kinase C inhibitors, which did not affect I
B degradation or nuclear translocation/DNA binding of NF
B. SB203580, a p38 MAPK inhibitor, completely inhibited TNF-induced synthesis of IL-6 and expression of a reporter gene containing a minimal promoter with two NF
B elements; however, neither TNF-induced DNA binding of NF
B nor TNF-induced phosphorylation of its subunits was modulated by SB203580 (52, 55), and other studies gave similar results (53, 56).
Several reports show that activation of kinase pathways are required for induction of NF
B-dependent activities, and these kinases include NIK/MAP3K (57), MAPK P38 and ERK (52, 55), MEKK1 (58), phosphatidylcholine-specific phospholipase C (51, 59) and protein kinase C (51, 60, 61, 62), tyrosine kinase (53), PKA (63), casein kinase II (CK II) (64), Akt/PKB (65, 66), and CaMKIV (21). The role of kinase activation by E2 through nongenomic pathways was investigated using a series of kinase inhibitors, and only inhibitors of Ca2+-dependent kinases (EGTA-AM and KN-93) block hormonal activation of pM-65c2 and p5310 (Fig. 7
, B and C). A recent study reported that E2 increases mobilization of intracellular Ca2+ in MCF-7 cells and this response has been linked to activation of the MAPK pathway (40). Results of inhibitory studies with the intracellular Ca2+ chelator EGTA-AM indicate that Ca2+ mobilization is also important for activation of p65 in MCF-7 cells (Fig. 7
, B and C); however, this response was not blocked by the MAPKK inhibitor PD98059. Intracellular Ca2+ also plays an important role in activation of Ca2+/CaM-dependent kinases (67), and a recent study showed that these kinases stimulate activation and phosphorylation of p65 in CV-1 and HeLa cells (21). It was reported that constitutively active CaMKI and CaMKIV (but not CaMKII) activated p65 and this response was inhibited by the Ca2+/CaM-dependent kinase inhibitor KN-93 (21). In contrast, our results show that constitutively active CaMKIV but not CaMKI activated p65 in MCF-7 cells (Fig. 7D
). Hormonal activation of both CaMK1 and CaMKIV was further investigated in MCF-7 cells, treated with [32P]-labeled orthophosphate, and the results showed that E2 induced phosphorylation of CaMKIV (Fig. 7E
), whereas phosphorylation of CaMKI in MCF-7 cells was not detected (data not shown). These data suggest that hormonal activation of p65/NF
B in MCF-7 cells is CaMKIV-dependent and related to increased mobilization of Ca2+ in this cell line (40). ChIP has identified nuclear ER
bound to the E2-responsive region of the p53 gene promoter (Fig. 4B
), and ER
can interact with p65, CTF-1 and Sp1 proteins (31, 32, 47, 68), which also bind elements within this region. Results of transactivation, gel mobility shift and kinase inhibitor assays suggest that p53 is activated by E2 through nongenomic pathways. However, the detection of ER
bound to the p53 promoter suggests that nuclear ER
may contribute to the observed responses and this is currently being investigated.
In summary, our results show that activation of p53 gene expression in MCF-7 cells by E2 is dependent on nongenomic activation of CaMKIV, which results in activation of p65. This pathway is consistent with previous reports showing that E2 activates release of intracellular calcium (40, 69, 70, 71) and CaMKIV stimulates NF
B through phosphorylation of p65 (21). Although E2 stimulates proliferation of MCF-7 cells, the concomitant induction of p53, a protein associated with growth inhibition, is not unexpected. For example, estrogen also induces proteasome-dependent degradation of ER
protein (72, 73, 74, 75) and expression of genes such as early growth response 1 (76) that can act as a negative transcription factor. p53 and BRCA1 are genes associated with inhibition of cell growth, DNA repair and apoptosis, and both BRCA1 and p53 are induced by E2 in breast cancer cells (18, 19, 20, 77, 78). Interestingly, both proteins physically interact with ER
and inhibit ER
-mediated transactivation (79, 80), suggesting that hormonal activation of p53 and BRCA1 may contribute to regulatory pathways that lead to inhibition of E2-induced cell growth and differentiation. Other mitogens and growth regulatory proteins also induce inhibitory pathways that temporally regulate the magnitude and duration of their responses (81, 82). The role of p53 as negative regulator of hormone-induced proliferation of MCF-7 cells and the mechanisms of E2-dependent nongenomic activation of calcium-dependent kinase pathways are being further investigated in this laboratory in ER-positive and ER-negative cancer cell lines.
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MATERIALS AND METHODS
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Cells, Chemicals, Biochemicals, and Other Materials
MCF-7 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA), and maintained in MEM media with phenol red and supplemented with 0.22% sodium bicarbonate, 10% fetal bovine serum (FBS), 0.011% sodium pyruvate, 0.1% glucose, 0.24% HEPES, 10-6% insulin and 10 ml/liter antibiotic solution (Sigma, St. Louis, MO). Cells were grown in 100-cm2 culture plates in an air:carbon dioxide (95:5) atmosphere at 37 C, and passaged every 6 d. Cells were seeded in phenol red free DME/F12 media with 5% charcoal-stripped FBS (CSS). DMSO, E2, PBS, and 100X antibiotic solution were purchased from Sigma. FBS was obtained from Intergen (Purchase, NY). STAT-60 RNA Extract Kit was purchased from Tel-Test "B", Inc. (Friendswood, TX). [32P]ATP (3000 Ci/mmol) and horseradish peroxidase- substrate for Western blot analysis were purchased from NEN Life Science Products (Boston, MA). [35S]Methionine and Hybond-N nylon membrane for Northern blot analysis were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Antibodies for p53 (sc-126), Sp1 (sc-59), CTF-1, Sp3 (sc-644), NF
B p65 (sc-109X), p50 (sc-1190X), I
B-
(sc-371), CaMKIV, CaMKI, and preimmune IgG were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and NF-YA antibody from Rockland Inc. (Gilbertsville, PA). Poly deoxy-(insolinic-cytidylic) acid, restriction enzymes, and T4-polynucleotide kinase, and pGL2 luc reporter vector, rabbit reticulocyte lysate system were purchased from Promega Corp. (Madison, WI) and Roche Molecular Biochemicals (Indianapolis, IN). Reporter Lysis Buffer and Luciferase Reagent for Luciferase studies were purchased from Promega Corp. ß-Galactosidase (ß-gal) Reagent was purchased from Tropix (Bedford, MA). Plasmid preparation kit was purchased from QIAGEN (Santa Clarita, CA). The mammalian MATCH MAKER two-hybrid assay kit was purchased from CLONTECH Laboratories, Inc. (Palo Alto, CA). All other chemicals and biochemicals were the highest quality available from commercial sources. Lab-Tek Chamber slides was purchased from Nalge Nunc International (Naperville, IL). InstantImager and LumiCount were purchased from Packard (Meriden, CT).
Oligonucleotides and Plasmids
The p53 cDNA probe was purchased from Geneka Biotechnology Inc. (Montréal, Québec, Canada). p53 promoter- derived oligonucleotides, primers employed in plasmid construction, and consensus oligonucleotides for EMSA were synthesized by Genosys/Sigma (The Woodlands, TX) or by the laboratory of Dr. James Derr (Department of Veterinary Pathobiology, TAMU). Consensus oligonucleotides used in EMSA competition experiments are Sp1 (5'-AGC TTA TTC GAT CGG GGC GGG GCG AGC g-3'), ERE (5'-GTC CAA AGT CAG GTC ACA GTG ACC TGA TCA AAG TT-3'), NF-Y (AGA CCG TAC GTG ATT GGT TAA TCT CTT), CTF-1 (TTT TGG ATT GAA GCC AAT ATG ATA A), LSF (GAG CAA GCA CAA ACC AGC CAA), NF
B (AGT TGA GGG GAC TTT CCC AGG C). All the sequences listed in this paper are sense strands. They might have been synthesized in both sense and anti-sense strands, annealed and phosphorylated when necessary. Some sequences may contain additional restriction sites at flanking sites for cloning, however the linkers are not indicated.
The p1 CAT construct containing the human p1 p53 promoter was kindly provided by Dr. David Reisman (University of South Carolina, Columbia, SC), and the p53 promoter fragments were PCR amplified with appropriate primers. The p53 promoter luc reporters were constructed using pGL2 (Promega Corp.) for cloning of p531 to -8, and TpGL2 for p539 to -14. The TpGL2 vector was constructed by inserting a minimal TATA sequence (GCT GTA GGG TAT ATA ATG GAT CA) with linker into the BglII and the HindIII sites in pGL2. The reverse primers used for constructing critical mutated reporter p5310 m2 was AGT CTC GAG CAC ATG GGA GGG Gag cTC aaC ggT CCC ATC AAC C. All ligation products were transformed into TOP10F' competent cells (Invitrogen, Carlsbad, CA). Plasmids were confirmed by restriction enzyme mapping and DNA sequencing. High-quality plasmids for transfection were prepared using QIAGEN Plasmid Megaprep Kit (QIAGEN). The human ER expression plasmid was kindly provided by Dr. Ming-jer Tsai (Baylor College of Medicine, Houston, TX). The dnNF-YA (Dr. Michael Schweizer, Institute of Food Research, Norwich Research Park, Colney, Norwich, UK), pPacP65 and pdI (Drs. Claus Scheidereit and Vigo Heissmeyer, Max-Delbruck-Centrum fur Molekulare Medizin, Berlin, Germany), I
B-SR (Drs. Albert Baldwin and Jacqueline Norris, University of North Carolina, Chapel Hill, NC), pCTF-1 (Dr. Robert Tjian, University of California, Berkeley, CA) and dnSp1 (Dr. Gerald Thiel, University of the Saarland Medical School, Hamburg, Germany) plasmids were generous gifts. Constitutively active CaMKIV and CaMKI were kindly provided by Dr. Eric Olson (University of Texas Southwestern Medical School, Dallas, TX) and Dr. Anthony Means (Duke University Medical School, Durham, NC), respectively. The Gal4 reporter containing 5X Gal4 DBD (pGAL4-luc) and p65c2 (previously named Gal4-p65
Sma) containing p65 C-terminal 30 amino acids were kindly provided by Dr. Marty Mayo (University of North Carolina, Chapel Hill, NC). The pM-p65 was constructed by inserting p65 cDNA sequence from pPacP65 into EcoRI and XbaI sites of pM (CLONTECH Laboratories, Inc.), and p65c1 is an EcoRI restriction of pM-p65. The pV-CTF-1 was constructed by inserting CTF-1 cDNA sequence from pCTF-1 into EcoRI and XbaI sites of pVP16 (CLONTECH Laboratories, Inc.).
Northern Blot Analysis
MCF-7 cells were seeded in 5% CSS DME/F12 media for 24 h, and in serum-free DME/F12 media for another 48 h. Cells were treated in fresh serum-free media and harvested at various times. Total RNA was isolated using STAT-6 Kit. Twenty micrograms of total RNA were diluted in 2x RNA sampling buffer (20% formaldehyde; 1.65% Na2HPO4, pH 6.8; 63.5% formamide; 1x loading buffer), and separated on 1.2% agarose gel with 1 M formaldehyde in 1x running buffer (20 mM Na2HPO4, 2 mM CDTA, pH 6.8). After transfer onto Hybond-N nylon membrane, and prehybridized and hybridized in NENhybe solution [750 mM NaCl, 50 mM NaH2PO4, 5 mM EDTA (pH 7.4); 10% dextran sulfate, 0.1% polyvinyl pyrolidine, 0.1% Ficoll, 0.1% BSA, 1% sodium dodecyl sulfate (SDS)] at 65 C without/with 32P-ATP-labeled 40-oligomer p53 cDNA probe for 16 h. The membrane was washed 15 min with 1x washing buffer containing 150 mM NaCl, 10 mM NaH2PO4, 1 mM EDTA (pH 7.4), and 2% SDS. Blots were visualized by autoradiography and bands were quantitated on InstantImager (Packard). The membrane was then stripped (0.1x saline sodium citrate, 1% SDS) and rehybridized with ß-tubulin probe as a control.
Transient Transfection and Luciferase Activity Assay
MCF-7 cells were seeded in 5% CSS DME/F12 media in 60-mm plate 1 d before transfection using calcium phosphate-DNA coprecipitation method. Reporter plasmid DNA (2.5 µg) and 1.0 µg ß-gal DNA, with or without 1.0 µg wild-type human ER DNA, were used for transfection in promoter analysis; 0.252.0 µg of dnNF-Y, pdI, I
B-SR, and dnSp1 were used for cotransfection, and their related empty vectors were added to balance total transfected DNA amounts; 2.5 µg GAL4-luc reporter, 0.25 µg pM or pVP16 and their related fusions were used in mammalian two-hybrid system transfection. After incubation for 1620 h, cells were washed with PBS and treated with E2 or DMSO (as control) for 24 h if applicable in fresh media. Cells were then lysed with 400 µl of 1x reporter lysis buffer. Thirty microliters of cell extract were subjected to luc and ß-gal assays. LumiCount was used to quantitate luc and ß-gal activities, and the luc activities were normalized to ß-gal activity.
Preparation of Cytosolic and Nuclear Extracts
MCF-7 cells were seeded in 5% CSS DME/F12 media in 150-mm plates for 2 d, and treated as indicated. Cells were then harvested in 3 ml of PBS. After spinning down, cells were resuspended in HED buffer (25 mM HEPES; 1.5 mM EDTA; 1.0 mM dithiothreitol, pH 7.6). After incubation on ice for 10 min, cells was pelleted by centrifuging at 2000 x g for 10 min. Supernatant was discarded, cells were lysed in HEGD buffer (25 mM HEPES; 1.5 mM EDTA; 1.0 mM dithiothreitol; 10% glycerol, pH 7.6), syringed with 21-gauge needles 6x, checked with trypan blue for cell breakage. Cytosolic protein (supernatant) was collected after centrifuging at 4000 x g for 15 min. Nuclei were then washed with HEGD buffer (5x), and lysed with HEGDK buffer (25 mM HEPES; 1.5 mM EDTA; 1.0 mM dithiothreitol; 10% glycerol; 500 mM KCl, pH 7.6) on ice for 60 min. Nuclear protein was collected after centrifuging at 12,000 x g for 30 min at 4 C, aliquoted and stored at -70 C.
Western Blot Analysis
MCF-7 cells were seeded in 5% CSS DME/F12 media for 2 d, and pretreated with CHX and treated with E2 or IL-1ß as indicated. Cytosolic and nuclear protein were collected, and protein samples were heated at 100 C for 5 min, separated on 10% SDS-PAGE at 160V for 3 h in 1x running buffer (25 mM Tris-HCl; 192 mM glycine; 0.1% SDS, pH 8.3), and transferred to polyvinylidenedifluoride membrane (Amersham Pharmacia Biotech) at 100 V for 2 h at 4 C in 1x transfer buffer (48 mM Tris-HCl, 39 mM glycine, 0.075% SDS). The polyvinylidenedifluoride membrane was blocked in 5% milk-TBS (10 mM Tris-HCl, 150 mM NaCl, pH8.0) with gentle shaking for 1 h, and incubated in fresh 5% milk-TBS with 1:3000 (for p53 and CaMKIV), 1:1000 (for I
B) or 1:4000 (for p65) primary antibody (Santa Cruz Biotechnology, Inc.) for 1 h with gentle shaking. After washing in 1x TBS for 5 min (3x), 1:4,000 secondary antibody in 5% milk-TBS was incubated with shaking for 1 h. The membrane was then washed again in TBS buffer for 1 h, incubated in 10 ml of chemiluminescent substrate (electrochemiluminescence) (NEN Life Science Products) for 1.0 min, and exposed to Kodak (Rochester, NY) X-Omat AR autoradiography film immediately. Band intensities were evaluated by scanning laser densitometry (Sharp Electronics Corp.) and background subtraction using Zero-D Scanalytics software (Scanalytics Corp.). The membrane was reprobed with Sp1 antibody as control.
Immunocytochemistry
MCF-7 cells were seeded in 5% CSS DME/F12 media to 50% confluency on Lab-Tek Chamber slides (Nalge Nunc International), treated with E2 or DMSO for various time and washed with 20 mM PBS. Slides were fixed for 10 min in -20 C methanol, air-dried, and washed in 0.3% Tween/PBS (Use 0.3% Tween in 20 mM PBS for all subsequent washing steps) for 5 min. The slides were then blocked with isotype IgG for 1 h. The NF
B p65 antibody (Santa Cruz Biotechnology, Inc.) was diluted in antibody dilution buffer (1% BSA in 0.3% Tween/PBS), and 1:750 p65 antibody was added to the well. Slides were incubated overnight at 4 C in a humid chamber to prevent evaporation of the antibody solution. After washing slides in 0.3% Tween/PBS for 10 min (3x), diluted second antibody (raised against the same species as the primary antibody) was added. The secondary antibody was directly conjugated with fluorochrome. Slides were incubated for 2 h at room temperature in a dark humid chamber, and then washed in 0.3% Tween/PBS for 10 min (4x), and rinsed in deionized water. Slides were mounted in glycerol/phenylenediamine and visualized. Samples without primary antibody or secondary antibody were also used as control.
Gel EMSA
Nuclear extracts were prepared from MCF-7 cells treated with 10 nM of E2 for 2 h utilizing cells maintained in MEM media supplemented with 10% FBS. Synthetic oligonucleotides were annealed, and labeled with T4-polynucleotide kinase and [
-32P]-ATP. EMSA was performed by incubating 35 µg of nuclear extract in 25 µl of binding buffer (25 mM HEPES; 1.5 mM EDTA; 1.0 mM dithiothreitol; 2 mM MgCl2; 10% glycerol; 100 mM KCl, pH 7.6) in the presence of 1 µg deoxy-(insolinic-cytidylic) at 4 C for 5 min. 32P-labeled oligonucleotide (50,000 cpm) was added and incubated for an additional 15 min at 25 C. Excess unlabeled DNA used for competition and antibodies used for supershifts were added before 32P-labeled oligonucleotides. For analysis of Sp1-DNA binding, 0.1 mM ZnCl2 was included in the incubation reaction. The reaction mixture was separated on 5% polyacrylamide gel (acrylamide:bisacrylamide, 30:0.8) at 140 V in 1x TBE buffer (0.09 M Tris-HCl; 0.09 M boric acid; and 2 mM EDTA, pH 8.3). The gel was dried and exposed to PhosphoScreen and visualized on STORM.
In Vivo Phosphorylation
MCF-7 cells were seeded in 100-mm dishes with 10% FBS MEM media at 30% confluency for 24 h, and cultured in 5% CSS DME/F12 media for another 24 h. Cells were then changed to serum-free DME/F12 and incubated with 200 µCi 32P-orthophosphate for 3 h. Cells were then treated with 10 nM E2 for the indicated times, and washed with PBS (3x). Whole cell lysate (WCL) was collected and immunoprecipitated with CaMKIV antibody. Protein bound beads were suspended in 1x Western sampling buffer, and heated at 100 C for 5 min. Protein samples were separated on 10% SDS-PAGE. The gel was dried and exposed to PhosphoScreen and quantitated on STORM.
Coimmunoprecipitation/In Vitro
For immunoprecipitation by antibodies against NF
B p65, in vitro transcribed/translated proteins of CTF-1 and NF
B were obtained using the rabbit reticulocyte lysate system (Promega Corp.). Protein was labeled using [35S]methionine (Amersham Pharmacia Biotech). Equal volumes of 10 µl of CTF-1 and NF
B proteins were incubated for 1 h at room temperature. To this mixture, NF
B p65 antibodies were added and samples were subsequently incubated for 2 h on ice. Protein G-agarose beads (Santa Cruz Biotechnology, Inc.) were added and incubated at 4 C for 35 h. Immunoprecipitation of protein G-bound proteins was conducted according to manufacturers recommendations and subsequently separated by 7% SDS-PAGE electrophoresis and visualized by autoradiography.
Coimmunoprecipitation/Western Blot
MCF-7 cells were seeded in 5% CSS DME/F12 media in 150-mm plates for 2 d, and treated as indicated. Cells were rinsed with PBS at room temperature, and harvested in 0.5 ml of HEGD buffer [1x PBS, 1% Nonidet P-40 (NP-40), 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride (PMSF)]. Cells were lysed by freeze-thawing in liquid nitrogen/ice water (3x) and brought to 0.5 M in sodium chloride by addition of 4 M sodium chloride, 20 mM HEPES (pH 7.4), then transferred using a syringe with a 21- gauge needle to a fresh tube, and incubated with 10 µl of 10 mg/ml PMSF for 3060 min on ice. Supernatant was collected as WCL after microcentrifuging at 10,000 x g for 10 min at 4 C. WCL was precleared by adding 0.25 µg of control IgG with 20 µl protein G-agarose conjugate to approximately 1 ml WCL, and incubating at 4 C for 30 min with shaking. Beads were pelleted by centrifuging at 1000 x g for 5 min at 4 C, and supernatant (cell lysate) was collected. To 200 µg of the supernatant, 2 µg of CTF-1 or normal IgG antibody was added. The reaction mixture was incubated at 4 C for 1 h, and mixed with 20 µl of resuspended protein A/G-agarose beads and incubated for 1 h on a rocker platform at 4 C. Pellet was collected by contrifugation at 1000 x g for 5 min at 4 C, and washed with HEGDK buffer (4x). After the final wash, the pellet was resuspended in 40 µl of 1x electrophoresis buffer for Western blot analysis using p65 antibodies as described above.
ChIP Assay
MCF-7 breast cancer cells were grown in 150-mm tissue culture plates to 8095% confluency and treated with 10 nM E2 for 15 min. Formaldehyde was then added to the medium to give a 1% solution and incubated with shaking for 10 min at 20 C. After addition of glycine (0.125 M) and incubation for 10 min, the media was removed, cells were washed with PBS and 1 mM PMSF, scraped, and collected by centrifugation. Cells were then resuspended in swell buffer (85 mM KCl, 0.5% NP-40, 1 mM PMSF, 5 µg/ml leupeptin and aprotinin at pH 8.0) and homogenized. Nuclei were isolated by centrifugation at 1500 x g for 30 sec, then resuspended in sonication buffer (1% SDS; 10 mM EDTA; 50 mM Tris, pH 8.1), and sonicated for 4560 sec to obtain chromatin with appropriate fragment lengths (5001000 bp). This extract was then centrifuged at 15,000 x g for 10 min at 0 C, aliquoted and stored at -70 C until used. The cross-linked chromatin preparations were diluted in buffer (1% Triton X; 100 mM NaCl; 0.5% SDS; 5 mM EDTA; and Tris, pH 8.1), and 20 µl of Ultralink protein A or G or A/G beads (Pierce Chemical Co., Rockford, IL) was added per 100 µl chromatin and incubated for 4 h at 4 C. A 100-µl aliquot was saved and used as the 100% input control. Salmon sperm DNA, specific antibodies, and 20 µl Ultralink beads were added and the mixture incubated for 6 h at 4 C. Samples were then centrifuged; beads were resuspended in dialysis buffer, vortexed for 5 min at 20 C, and centrifuged at 15,000 x g for 10 sec. Beads were then resuspended in immunoprecipitation buffer (11 mM Tris; 500 mM LiCl; 1% NP-40; 1% deoxycholic acid, pH 8.0) and vortexed for 5 min at 20 C. The procedures with the dialysis and immunoprecipitation buffers were repeated (34 times), and beads were then resuspended in elution buffer (50 nM NaHCO3, 1% SDS, 1.5 µg/m sonicated salmon sperm DNA), vortexed, incubated at 65 C for 15 min. Supernatants were then isolated by centrifugation and incubated at 65 C for 6 h to reverse protein-DNA cross-links. Wizard PCR kits (Promega Corp.) were used for additional DNA cleanup and PCR using [32P]
-deoxy-CTP was used to detect the presence of promoter regions immunoprecipitated with commercially available CREB1, p65, CTF-1, ER
, or Sp1 antibodies (Santa Cruz Biotechnology, Inc.). An improved ChIP assay using an increased amount of cross-linked chromatin for the immunoprecipitation assay was carried out as described (28). The same p53-specific primers were used in a two-step PCR reaction (9560 C). PCR products were run on a 2% agarose gel and stained with ethidium bromide. The following primers were used for PCR analysis of immunoprecipitated promoter regions:
p53: Fw (-196), 5'-GCG GTA CCC CAG GTC GGC GAG AAT CC-3'; Rv (-6), 5'-GGG CTC GAG TCT AGA CTT TTG AGA AGC-3'.
Cathepsin D: Fw (-294), 5'-TCC AGA CAT CCT CTC TGG AA-3'; Rv (-54, 5'-GGA GCG GAG GGT CCA TTC-3'.
Cathepsin D (exon 2): Fw (+2469), 5'-TGC ACA AGT TCA CGT CCA TC-3'; Rv (+2615), 5'-TGT AGT TCT TGA GCA CCT CG-3'.
Statistical Analysis
Statistical differences between different groups were determined by ANOVA and Scheffés test for significance. The data for all transient transfection studies are presented as mean ± SD for at least three replicate determinations for each treatment group and were observed in at least two sets of experiments.
 |
FOOTNOTES
|
---|
The financial assistance of the NIH (ES09253 and ES09106) and the Texas Agricultural Experiment Station is gratefully acknowledged.
Abbreviations: CaMKIV, Calmodulin kinase IV; CAT, chloramphenicol acetyltransferase; ChIP,Chromatin immunoprecipitation; CHX, cycloheximide; CREB, cAMP response element binding protein; CSS, charcoal-stripped FBS; CTF-1, CCAAT-binding transcription factor-1; DBD, DNA binding domain; DMSO, dimethylsulfoxide; dn, dominant negative; E2, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen-responsive element; FBS, fetal bovine serum; ß-gal, ß-galactosidase; I
B-SR, I
B super repressor; luc, luciferase; NF
B, nuclear factor
B; NF-Y, nuclear factor-Y; NP-40, Nonidet P-40; pdI, precursor-derived inhibitor; PMSF, phenylmethylsulfonylfluoride; SDS, sodium dodecyl sulfate; WCL, whole cell lysate.
Received for publication January 7, 2002.
Accepted for publication May 10, 2002.
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