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by DNA Methyltransferase and Histone Deacetylase Inhibition in Human ER--negative Breast Cancer Cells1
The Johns Hopkins Oncology Center, Johns Hopkins University, Baltimore, Maryland 21231
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ABSTRACT |
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-negative human breast cancer cells with the DNMT inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) led to ER mRNA and protein re-expression. Also, the HDAC inhibitor trichostatin A (TSA) could induce ER transcript about 5-fold. Here we show that 5-aza-dC alone induced ER transcript about 30–40-fold, and the addition of TSA elevated ER mRNA expression about 10-fold more in the human ER-negative breast cancer cell lines MDA-MB-231 and MDA-MB-435. Overall, the combination of 5-aza-dC and TSA induced a 300–400-fold increase in ER transcript. Restoration of estrogen responsiveness was demonstrated by the ability of the induced ER protein to elicit estrogen response element-regulated reporter activity from an exogenous plasmid as well as induce expression of the ER target gene, progesterone receptor. The synergistic activation of ER occurs concomitantly with markedly reduced soluble DNMT1 expression and activity, partial demethylation of the ER CpG island, and increased acetylation of histones H3 and H4. These data suggest that the activities of both DNMT1 and HDAC are key regulators of methylation-mediated ER gene silencing. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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50% of unselected primary breast cancers and most ER-negative breast cancer cell lines (7)
. The methylation of these CpG cluster sites is associated with either reduced or absent ER expression. The DNMT inhibitor 5-aza-dC is widely used to study the reexpression of genes silenced by promoter methylation (8) . 5-aza-dC exerts its demethylating function through sequestration of DNMT1 to 5-aza-dC-substituted DNA by the irreversible binding of the cysteine in the catalytic domain of the DNMT1 enzyme to the 6 position of the cytidine ring (9 , 10) . In our previous study, treatment of ER-negative human breast cancer cells with the methyltransferase inhibitors 5-aza-cytidine or 5-aza-dC led to partial demethylation of the ER CpG island, reexpression of ER mRNA, and synthesis of functional ER protein. Restoration of ER function was documented by eliciting ERE-driven promoter activity from an exogenous plasmid as well as the expression of the ER-responsive gene, PR (11) .
Inactive chromatin built on methylated CpG clusters is emerging as an important molecular mechanism to silence a variety of methylated tissue-specific and imprinted genes (12 , 13) . This process involves methyl CpG-binding proteins to recruit HDAC family members. These HDACs remove acetyl groups from lysine residues of core histones, particularly H3 and H4, to increase ionic interactions between positively charged lysines of histones and negatively charged DNA, thereby generating a more compact nucleosome structure that limits gene activity. In our previous study, TSA, a potent and reversible HDAC inhibitor, could induce ER mRNA by 5-fold. This transcriptional activation is associated with increased sensitivity to DNase I at the ER locus without alteration of the methylated CpG sites, suggesting that an accessible chromatin structure is important in ER expression (6) .
Recently, in vitro studies have shown that DNMT1 interacts physically with either HDAC1 or HDAC2 (14 , 15) , in addition to its ability to methylate hemimethylated CpG sites in DNA. These findings suggested that the ability of DNMT1 to repress transcription after replication through its regulatory NH2 terminus is partially dependent on HDAC activity. A study using 5-aza-dC followed by TSA treatment of human colon cancer and leukemia cells robustly reactivated multiple methylated genes such as MLH1, TIMP3, CDK2B, and CDK2A. This finding supports the essential roles of both DNMT and HDAC in silencing expression of endogenous methylated genes (16) .
Here we show that cotreatment with DNMT and HDAC inhibitors can synergistically induce ER gene expression in ER-negative breast cancer cells. Induced expression of ER mRNA and protein is associated with expected ER function on estrogen-responsive targets. First, ERE-driven reporter activity from an exogenous plasmid is induced by the treatments and blocked by the ER antagonist ICI 182,780. Secondly, the treatments stimulate expression of the ER-responsive gene, PR, which is again suppressed by ICI 182,780. Our data show that synergistic induction of ER occurs in conjunction with reduced soluble DNMT1 expression and DNMT activity, partial demethylation of the ER CpG island, and increased acetylation of histones H3 and H4. These data suggest that pharmacological intervention against both DNMT and HDAC can synergistically reactivate the methylated ER gene expression and restore ER function.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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antibody, 1D5, was obtained from Coulter Immunotech (Marseille, France). The pure antiestrogen ICI 182,780 was a generous gift from AstraZeneca Pharmaceuticals (Macclesfield, United Kingdom). For treatment, cells were seeded at a density of 5 x 105 cells/100-mm tissue culture dish. After 24 h of incubation, the culture media were changed to media containing 5-aza-dC for up to 96 h or TSA for 12 h. For the combination study, 5-aza-dC was present in culture for 96 h, and TSA was added for the last 12 h.
RNA Isolation, RT-PCR Analysis of ER and PR, and Quantitative Competitive PCR Analysis of ER.
Total cellular RNA was extracted by Trizol reagent (Life Technologies, Inc., Rockville, MD). RT-PCR was carried out according to our previously described method (6)
. RNAs under comparison were simultaneously reversibly transcribed to achieve equal efficiency for reverse transcription. ER mRNA expression was initially screened by RT-PCR. If ER mRNA was present, a previously reported quantitative competitive PCR assay was performed to determine the level of ER transcript in treated MDA-MB-231 and MDA-MB-435 cells as compared with the expression level in untreated cells or the control ER-positive MCF-7 cells (6)
. PR RT-PCR primers were described previously (11)
, and the PCR reaction was performed with an Advantage GC2 PCR kit (Clontech, Palo Alto, CA) according to the protocol recommended by the supplier.
Genomic DNA Isolation and MSP Analysis of ER CpG Island.
DNA was isolated by standard phenol-chloroform extraction. Isolated DNA was subjected to modification by sodium bisulfite to convert unmethylated cytosines but not methylated cytosines to uracils as described previously (19)
. Methylation status of the bisulfite-modified DNA at the ER locus was characterized by MSP using our previously reported method (7)
.
Western Blot Analysis of ER, PCNA, or DNMT1 Expression.
Proteins from detergent-lysed cells were quantified by using the BCA protein assay kit (Pierce, Rockford, IL). The Western blot procedure was reported previously (20)
. Briefly, after protein separation by electrophoresis and transfer to nitrocellulose, blots were probed for 1 h with 2 µg/ml either mouse monoclonal antibody 1D5 (ER specific) or Ab-1 (PCNA specific) or affinity-purified rabbit polyclonal IgG (DNMT1 specific) at a 1:1000 dilution in PBS containing 5% dry milk. After incubation with peroxidase-labeled secondary rabbit antimouse or goat antirabbit antibodies, the immunoreactive proteins were detected by the enhanced chemiluminescence method as recommended by the manufacturer (Amersham Pharmacia Biotech, Piscataway, NJ). Protein band intensities were quantified by densitometry (EagleSight Software of Eagle Eye II Imaging System; Stratagene, La Jolla, CA).
DNMT Activity Assay.
Lysates from cells growing in the presence or absence of 5-aza-dC, TSA, or 5-aza-dC and TSA were prepared and assayed for enzyme activity by the incorporation of [3H]methyl-S-adenosylmethionine into poly(dI-dC:dI-dC) as described previously (21)
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Analysis of Acetylated Histones.
Cellular histone extraction was performed using 2 x 106 cells according to the procedure of Yoshida et al. (22)
, with the following modifications. The acid (H2SO4)-soluble supernatant was precipitated with 10 volumes of cold acetone. After overnight precipitation, histones were collected by centrifugation. The pellet containing histones was dissolved in 50 µl of H2O, and proteins were quantified by using the BCA protein assay kit.
The extracted histones were separated by electrophoresis on a 15% SDS-PAGE gel and transferred to nitrocellulose membrane. After sequential incubations with rabbit anti-acetylated H3 and H4 polyclonal sera and goat antirabbit horseradish peroxidase-conjugated secondary antibody, the immunoreactive proteins were detected by the enhanced chemiluminescence method.
Transfections and Luciferase Assays.
The ERE2-tk-luciferase/SV-neo plasmid has been described previously (11)
. MDA-MB-231 cells were transfected with the plasmid using LipofectAMINE reagent and selected with G418 (1 mg/ml; Life Technologies, Inc.) for 2 weeks. Isolated colonies of G418-resistant cells were pooled and used for analysis. The pooled, transfected cells were maintained in culture media with 1 mg/ml G418. Luciferase activities were determined by Bright-Glo Luciferase Assay System (Promega, Madison, WI), and the activities were normalized for protein concentrations.
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Results and Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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, ER mRNA was induced 31-fold by treatment with 5-aza-dC for 96 h as compared with vehicle-treated cells, whereas treatment with TSA alone for 12 h had little effect. The combined treatment led to a 400-fold induction in ER transcript. Thus, the brief addition of TSA to optimal 5-aza-dC treatment increased ER mRNA by about 13-fold, suggesting a synergistic effect.
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, treatment with 5-aza-dC alone induced ER mRNA by 36-fold, whereas treatment with TSA alone for 12 h had little effect. The combined treatment led to a 318-fold induction of ER transcript compared with that of vehicle-treated MDA-MB-435 cells (Fig. 1B)
. Thus, two different ER-negative cell lines showed evidence of synergistic reactivation of ER by the combined approach of DNMT and HDAC inhibitors.
We next studied how the levels of ER reactivation compared with endogenous ER expression in the human breast cancer cell line MCF-7. As shown in Fig. 1C
, 5-aza-dC induced ER transcripts in MDA-MB-231 and MDA-MB-435 cells to 4% and 2%, respectively, of the expression level found in MCF-7 cells. The addition of TSA to the 5-aza-dC-treated cultures induced ER mRNA to 50% and 20% of the MCF-7 expression levels, respectively. Thus, the combination treatment consistently and synergistically induced ER transcript in ER-negative breast cancer cell lines, supporting our hypothesis that the activities of both DNMT and HDAC are key regulators in ER gene silencing. The synergistic effect with an optimal exposure to DNMT inhibitor and a suboptimal exposure to HDAC inhibitor suggested a layered action on ER activation.
However, the combination of DNMT and HDAC inhibitors apparently did not reactivate ER mRNA expression to the levels seen in MCF-7 cells. This finding could reflect experimental variation or differences in genotypes between cell lines. Alternatively, it is possible that other mechanisms might also play a role in the silencing process. For example, other transcriptionally repressive complexes such as methyl-CpG binding protein/HDAC could also be involved in the silencing process. Also, some corepressor proteins in various repressive complexes are partially HDAC independent. For instance, a component of the repression mediated by the MeCP2 transcriptional repression domain is partially HDAC independent, and mSin3A could retain some ability to repress transcription, even in the absence of associated HDACs (12) . Also, DMAP-1, a corepressor in the DNMT1/HDAC gene transcriptional repression complex, is HDAC independent (14) . Therefore, simultaneous inhibition of several components in the methylation-associated repressive complexes might be necessary to achieve maximal reactivation of repressed genes.
This effect appeared to be relatively specific for the methylated ER gene because the treatments (alone or in combination) had no apparent effect on the level of expression of the unmethylated housekeeping gene, ß-actin, using a competitive quantitative PCR assay (data not shown). However, the combined treatment of the ER-negative breast cancer cells did reactivate expression of other genes silenced by methylation such as RARß2 or cyclin D2 (data not shown) in addition to ER.
Next, we investigated whether the activated ER transcript was translated into ER protein by Western analysis. The ER protein signal in 5-aza-dC-treated MDA-MB-231 cells was compared with that in the combination-treated cells after normalizing with ß-actin protein. As shown in Fig. 1D
, 5-aza-dC treatment of MDA-MB-231 cells reactivated the Mr 67,000 ER protein, whereas TSA had little effect. The amount of ER protein in combination-treated cells was about twice that in cells treated with 5-aza-dC alone. These data provide further evidence that ER gene expression was enhanced by the combined treatment with 5-aza-dC and TSA as compared with that seen with either drug alone.
Analyses of Activated ER Function in MDA-MB-231 Cells.
The ability of the re-expressed ER protein to mediate an estrogen response was tested next. For this study, a plasmid that contains a firefly luciferase gene under the transcriptional regulation of two EREs driving a thymidine kinase promoter was stably transfected into MDA-MB-231 cells. These cells were treated with 5-aza-dC or TSA alone and with the combination of both (see "Materials and Methods") in estrogen-containing culture media. As shown in Fig. 2A
, treatment with 5-aza-dC alone led to a 1.4-fold increase in promoter activity. TSA alone induced the reporter activity by 5-fold, an unexpected finding because 12 h of TSA induced little ER mRNA or protein. Possibly, this is due to the chromatin remodeling effect of TSA instead of ER involvement. Nevertheless, cotreatment with 5-aza-dC and TSA elicited a more than additive induction of reporter activity (about 8-fold; Fig. 2A
). Thus, the combination treatment enhanced ERE-driven reporter transcription in MDA-MB-231 cells when compared with single agents.
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, ICI 182,780 suppressed 5-aza-dC-stimulated ERE-mediated reporter activity but had no effect on TSA-mediated transcription. These results indicate that 5-aza-dC-activated reporter transcription (5-aza-dC alone or the combination) is a specific hormone effect and suggest that the effect of treatment by TSA alone on this reporter activity is not completely related to estrogen response. Indeed, the exact nature of the effect of TSA on this reporter needs to be further explored.
We further investigated the ability of the drug-induced ER to activate expression of the endogenous ER-responsive gene, PR. As shown in Fig. 2B
, treatment of MDA-MB-231 cells with 5-aza-dC or the combination induced PR mRNA expression, whereas ICI 182,780 or TSA alone had little or no effect. Cotreatment of MDA-MB-231 cells with 5-aza-dC/ICI 182,780 suppressed PR expression. Similarly ICI 182,780 treatment of cells growing in 5-aza-dC and TSA led to a marked decrease in PR expression. Together, these data suggest that drug-induced ER is functionally active.
Changes in DNMT1 Expression, ER CpG Island Methylation, and Increased H3 and H4 Acetylation Introduced by DNMT and HDAC Inhibitors.
To study the underlying mechanisms associated with the synergistic activation of the methylated ER gene, we examined the expression and activity of the methylation maintenance enzyme, DNMT1. The results in Fig. 3A
showed that addition of 2.5 µM 5-aza-dC to the culture of MDA-MB-231 cells for 96 h markedly depleted soluble DNMT1 protein expression and total DNMT activity from the whole cell protein lysates (Fig. 3B)
. Interestingly, TSA also down-regulated soluble DNMT1 protein expression with some reduction in DNMT activity. Down-regulation of DNMT1 expression by TSA was consistently obtained in several independent experiments. The combination treatment further reduced the amount of DNMT1 protein and total DNMT activity. Although the effect of 5-aza-dC on soluble DNMT1 protein and total DNMT activity was expected because it sequesters DNMTs after its incorporation into genomic DNA and inhibits its enzyme activity (10)
, the effect of TSA was not. One possible explanation is that the effect of TSA on DNMT1 expression and DNMT activity reflects its ultimate antiproliferative effects. A second explanation is that a possible functional or physical interaction exists between DNMT1 and HDAC. Because the cell proliferation marker PCNA interacts physically with DNMT1 (24)
, we examined its expression by immunoblot. As shown in Fig. 3A
, PCNA expression was not obviously modulated by any treatment. Thus, our data suggest a possible functional connection between DNMT1 and HDAC or physical dependency for protein stabilities because no obvious down-regulation of DNMT transcript was observed by the various treatments (data not shown). However, the discrepancy between the level of DNMT1 depletion and reduction in total DNMT activity induced by TSA could also reflect a role of other DNMT family members that are not affected by TSA.
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. The ER CpG island remained methylated in MDA-MB-231 cells treated with vehicle or TSA (100 ng/ml for 12 h). However, treatment with 5-aza-dC alone or in combination with TSA led to partial demethylation of the CpG island, in conjunction with ER gene re-expression. These data are consistent with our previous findings and reaffirm that DNA methylation is a participant in the regulation of ER gene expression (11)
.
We then studied whether HDAC inhibition directly acetylates histones and whether the increased histone acetylation is associated with ER gene activation. As shown in Fig. 3D
, the HDAC inhibitor TSA up-regulated H3 and H4 acetylation as expected, although the magnitude of ER gene activation was not clearly proportional to the degree of histone H3 and H4 acetylation.
In summary, a demethylated ER promoter sequence with increased histone H3 and H4 acetylation by sequential inhibition of both DNMT and HDAC is correlated with the synergistic activation of the ER gene. These data indicate that pharmacological intervention against DNMT and HDAC can lead to alteration of ER gene methylation and chromatin conformation change characterized by hyperacetylated histones. Future studies using coimmunoprecipitation or chromatin-immunoprecipitation assay to elucidate the individual components that form the transcriptionally repressive complex at the ER gene locus will be pursued, and the effects of pharmacological manipulation will be further explored.
Because ER is a critical growth-regulatory gene in breast cancer, it is important to better understand its transcriptional regulation. Our data suggest that a combination drug regimen of both DNMT and HDAC inhibitors can synergistically activate functional ER protein. Additional studies will be necessary to evaluate the clinical implications of this finding with regard to sensitivity to selective ER modulators such as tamoxifen or ICI 182,780.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grants CA78352 and P50CA88843 (to N. E. D.) and Department of Defense Breast Cancer Program Grant DAMD17-00-1-0301 (to X. Y.). 
2 Present address: National Cancer Institute, NIH, Bethesda, MD 20889.
3 Present address: Molecular Devices Corp., Sunnyvale, CA 94089.
4 To whom correspondence should be addressed, at Johns Hopkins University School of Medicine, 1650 Orleans Street, CRB Room 409, Baltimore, MD 21231. E-mail: davidna{at}jhmi.edu
5 The abbreviations used are: ER, estrogen receptor; 5-aza-dC, 5-aza-2'-deoxycytidine; DNMT, DNA methyltransferase; ERE, estrogen response element; HDAC, histone deacetylase; MSP, methylation-specific PCR; PR, progesterone receptor; TSA, trichostatin A; PCNA, proliferating cell nuclear antigen; RT-PCR, reverse transcription-PCR.
Received 6/ 6/01. Accepted 8/13/01.
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in human breast cancer cells by histone deacetylase inhibition. Cancer Res., 60: 6890-6894, 2000.This article has been cited by other articles:
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D. Sharma, J. Blum, X. Yang, N. Beaulieu, A. R. Macleod, and N. E. Davidson Release of Methyl CpG Binding Proteins and Histone Deacetylase 1 from the Estrogen Receptor {alpha} (ER) Promoter upon Reactivation in ER-Negative Human Breast Cancer Cells Mol. Endocrinol., July 1, 2005; 19(7): 1740 - 1751. [Abstract] [Full Text] [PDF]
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D. C. Drummond, C. Marx, Z. Guo, G. Scott, C. Noble, D. Wang, M. Pallavicini, D. B. Kirpotin, and C. C. Benz Enhanced Pharmacodynamic and Antitumor Properties of a Histone Deacetylase Inhibitor Encapsulated in Liposomes or ErbB2-Targeted Immunoliposomes Clin. Cancer Res., May 1, 2005; 11(9): 3392 - 3401. [Abstract] [Full Text] [PDF]
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 P. W. Laird Cancer epigenetics Hum. Mol. Genet., April 15, 2005; 14(suppl_1): R65 - R76. [Abstract] [Full Text] [PDF]
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 J G Moggs, T C Murphy, F L Lim, D J Moore, R Stuckey, K Antrobus, I Kimber, and G Orphanides Anti-proliferative effect of estrogen in breast cancer cells that re-express ER{alpha} is mediated by aberrant regulation of cell cycle genes J. Mol. Endocrinol., April 1, 2005; 34(2): 535 - 551. [Abstract] [Full Text] [PDF]
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Y. Xiong, S. C. Dowdy, K. C. Podratz, F. Jin, J. R. Attewell, N. L. Eberhardt, and S.-W. Jiang Histone Deacetylase Inhibitors Decrease DNA Methyltransferase-3B Messenger RNA Stability and Down-regulate De novo DNA Methyltransferase Activity in Human Endometrial Cells Cancer Res., April 1, 2005; 65(7): 2684 - 2689. [Abstract] [Full Text] [PDF]
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N. P. Mongan and L. J. Gudas Valproic acid, in combination with all-trans retinoic acid and 5-aza-2'-deoxycytidine, restores expression of silenced RAR{beta}2 in breast cancer cells Mol. Cancer Ther., March 1, 2005; 4(3): 477 - 486. [Abstract] [Full Text] [PDF]
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R. P. Danam, S. R. Howell, T. P. Brent, and L. C. Harris Epigenetic regulation of O6-methylguanine-DNA methyltransferase gene expression by histone acetylation and methyl-CpG binding proteins Mol. Cancer Ther., January 1, 2005; 4(1): 61 - 69. [Abstract] [Full Text] [PDF]
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R I Nicholson, C Staka, F Boyns, I R Hutcheson, and J M W Gee Growth factor-driven mechanisms associated with resistance to estrogen deprivation in breast cancer: new opportunities for therapy Endocr. Relat. Cancer, December 1, 2004; 11(4): 623 - 641. [Abstract] [Full Text] [PDF]
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 S. Guo and G. E. Sonenshein Forkhead Box Transcription Factor FOXO3a Regulates Estrogen Receptor Alpha Expression and Is Repressed by the Her-2/neu/Phosphatidylinositol 3-Kinase/Akt Signaling Pathway Mol. Cell. Biol., October 1, 2004; 24(19): 8681 - 8690. [Abstract] [Full Text] [PDF]
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P. Parrella, M. L. Poeta, A. P. Gallo, M. Prencipe, M. Scintu, A. Apicella, R. Rossiello, G. Liguoro, D. Seripa, C. Gravina, et al. Nonrandom Distribution of Aberrant Promoter Methylation of Cancer-Related Genes in Sporadic Breast Tumors Clin. Cancer Res., August 15, 2004; 10(16): 5349 - 5354. [Abstract] [Full Text] [PDF]
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J. J. Pinzone, H. Stevenson, J. S. Strobl, and P. E. Berg Molecular and Cellular Determinants of Estrogen Receptor {alpha} Expression Mol. Cell. Biol., June 1, 2004; 24(11): 4605 - 4612. [Full Text] [PDF]
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M. Widschwendter, K. D. Siegmund, H. M. Muller, H. Fiegl, C. Marth, E. Muller-Holzner, P. A. Jones, and P. W. Laird Association of Breast Cancer DNA Methylation Profiles with Hormone Receptor Status and Response to Tamoxifen Cancer Res., June 1, 2004; 64(11): 3807 - 3813. [Abstract] [Full Text] [PDF]
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 S. Barnes Soy Isoflavones--Phytoestrogens and What Else? J. Nutr., May 1, 2004; 134(5): 1225S - 1228S. [Abstract] [Full Text] [PDF]
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 M. P. Waalkes, J. Liu, H. Chen, Y. Xie, W. E. Achanzar, Y.-S. Zhou, M.-L. Cheng, and B. A. Diwan Estrogen Signaling in Livers of Male Mice With Hepatocellular Carcinoma Induced by Exposure to Arsenic In Utero J Natl Cancer Inst, March 17, 2004; 96(6): 466 - 474. [Abstract] [Full Text] [PDF]
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I. Girault, S. Tozlu, R. Lidereau, and I. Bieche Expression Analysis of DNA Methyltransferases 1, 3A, and 3B in Sporadic Breast Carcinomas Clin. Cancer Res., October 1, 2003; 9(12): 4415 - 4422. [Abstract] [Full Text] [PDF]
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 K. De Bosscher, W. Vanden Berghe, and G. Haegeman The Interplay between the Glucocorticoid Receptor and Nuclear Factor-{kappa}B or Activator Protein-1: Molecular Mechanisms for Gene Repression Endocr. Rev., August 1, 2003; 24(4): 488 - 522. [Abstract] [Full Text] [PDF]
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G. Graziani, L. Tentori, I. Portarena, M. Vergati, and P. Navarra Valproic Acid Increases the Stimulatory Effect of Estrogens on Proliferation of Human Endometrial Adenocarcinoma Cells Endocrinology, July 1, 2003; 144(7): 2822 - 2828. [Abstract] [Full Text] [PDF]
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P. J. Durda, I. S. Dunn, L. B. Rose, D. Butera, E. M. Benson, F. Pandolfi, and J. T. Kurnick Induction of "Antigen Silencing" in Melanomas by Oncostatin M: Down-Modulation of Melanocyte Antigen Expression Mol. Cancer Res., April 1, 2003; 1(6): 411 - 419. [Abstract] [Full Text] [PDF]
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D. Medina, F. S. Kittrell, A. Shepard, A. Contreras, J. M. Rosen, and J. Lydon Hormone Dependence in Premalignant Mammary Progression Cancer Res., March 1, 2003; 63(5): 1067 - 1072. [Abstract] [Full Text] [PDF]
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K. Ghoshal, J. Datta, S. Majumder, S. Bai, X. Dong, M. Parthun, and S. T. Jacob Inhibitors of Histone Deacetylase and DNA Methyltransferase Synergistically Activate the Methylated Metallothionein I Promoter by Activating the Transcription Factor MTF-1 and Forming an Open Chromatin Structure Mol. Cell. Biol., December 1, 2002; 22(23): 8302 - 8319. [Abstract] [Full Text] [PDF]
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C. M. Klinge, S. C. Jernigan, and K. E. Risinger The Agonist Activity of Tamoxifen Is Inhibited by the Short Heterodimer Partner Orphan Nuclear Receptor in Human Endometrial Cancer Cells Endocrinology, March 1, 2002; 143(3): 853 - 867. [Abstract] [Full Text] [PDF]
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