FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2100392901> ?p ?o ?g. }

• W2100392901 endingPage "2597" @default.
• W2100392901 startingPage "2585" @default.
• W2100392901 abstract "The aromatase gene encodes the key enzyme for estrogen formation. Aromatase enzyme inhibitors eliminate total body estrogen production and are highly effective therapeutics for postmenopausal breast cancer. A distal promoter (I.4) regulates low levels of aromatase expression in tumor-free breast adipose tissue. Two proximal promoters (I.3/II) strikingly induce in vivo aromatase expression in breast fibroblasts surrounding malignant cells. Treatment of breast fibroblasts with medium conditioned with malignant breast epithelial cells (MCM) or a surrogate hormonal mixture (dibutyryl (Bt2)cAMP plus phorbol diacetate (PDA)) induces promoters I.3/II. The mechanism of promoter-selective expression, however, is not clear. Here we reported that sodium butyrate profoundly decreased MCM- or Bt2cAMP + PDA-induced promoter I.3/II-specific aromatase mRNA. MCM, Bt2cAMP + PDA, or sodium butyrate regulated aromatase mRNA or activity only via promoters I.3/II but not promoters I.1 or I.4 in breast, ovarian, placental, and hepatic cells. Mechanistically, recruitment of phosphorylated ATF-2 by a CRE (–211/–199, promoter I.3/II) conferred inductions by MCM or Bt2cAMP + PDA. Chromatin immunoprecipitation-PCR and immunoprecipitation-immunoblotting assays indicated that MCM or Bt2cAMP + PDA stabilized a complex composed of phosphorylated ATF-2, C/EBPβ, and cAMP-response element-binding protein (CREB)-binding protein in the common regulatory region of promoters I.3/II. Overall, histone acetylation patterns of promoters I.3/II did not correlate with sodium butyrate-dependent silencing of promoters I.3/II. Sodium butyrate, however, consistently disrupted the activating complex composed of phosphorylated ATF-2, C/EBPβ, and CREB-binding protein. This was mediated, in part, by decreased ATF-2 phosphorylation. Together, these findings represent a novel mechanism of sodium butyrate action and provide evidence that aromatase activity can be ablated in a signaling pathway- and cell-specific fashion. The aromatase gene encodes the key enzyme for estrogen formation. Aromatase enzyme inhibitors eliminate total body estrogen production and are highly effective therapeutics for postmenopausal breast cancer. A distal promoter (I.4) regulates low levels of aromatase expression in tumor-free breast adipose tissue. Two proximal promoters (I.3/II) strikingly induce in vivo aromatase expression in breast fibroblasts surrounding malignant cells. Treatment of breast fibroblasts with medium conditioned with malignant breast epithelial cells (MCM) or a surrogate hormonal mixture (dibutyryl (Bt2)cAMP plus phorbol diacetate (PDA)) induces promoters I.3/II. The mechanism of promoter-selective expression, however, is not clear. Here we reported that sodium butyrate profoundly decreased MCM- or Bt2cAMP + PDA-induced promoter I.3/II-specific aromatase mRNA. MCM, Bt2cAMP + PDA, or sodium butyrate regulated aromatase mRNA or activity only via promoters I.3/II but not promoters I.1 or I.4 in breast, ovarian, placental, and hepatic cells. Mechanistically, recruitment of phosphorylated ATF-2 by a CRE (–211/–199, promoter I.3/II) conferred inductions by MCM or Bt2cAMP + PDA. Chromatin immunoprecipitation-PCR and immunoprecipitation-immunoblotting assays indicated that MCM or Bt2cAMP + PDA stabilized a complex composed of phosphorylated ATF-2, C/EBPβ, and cAMP-response element-binding protein (CREB)-binding protein in the common regulatory region of promoters I.3/II. Overall, histone acetylation patterns of promoters I.3/II did not correlate with sodium butyrate-dependent silencing of promoters I.3/II. Sodium butyrate, however, consistently disrupted the activating complex composed of phosphorylated ATF-2, C/EBPβ, and CREB-binding protein. This was mediated, in part, by decreased ATF-2 phosphorylation. Together, these findings represent a novel mechanism of sodium butyrate action and provide evidence that aromatase activity can be ablated in a signaling pathway- and cell-specific fashion. Inhibitors of the aromatase enzyme are currently the most effective and most commonly used noncytotoxic therapeutic compounds in estrogen receptor-positive postmenopausal breast cancer (1Coombes R.C. Hall E. Gibson L.J. Paridaens R. Jassem J. Delozier T. Jones S.E. Alvarez I. Bertelli G. Ortmann O. Coates A.S. Bajetta E. Dodwell D. Coleman R.E. Fallowfield L.J. Mickiewicz E. Andersen J. Lonning P.E. Cocconi G. Stewart A. Stuart N. Snowdon C.F. Carpentieri M. Massimini G. Bliss J.M. Intergroup Exemestane StudyN. Engl. J. Med. 2004; 350: 1081-1092Crossref PubMed Scopus (1675) Google Scholar, 2Goss P.E. Ingle J.N. Martino S. Robert N.J. Muss H.B. Piccart M.J. Castiglione M. Tu D. Shepherd L.E. Pritchard K.I. Livingston R.B. Davidson N.E. Norton L. Perez E.A. Abrams J.S. Therasse P. Palmer M.J. Pater J.L. N. Engl. J. Med. 2003; 349: 1793-1802Crossref PubMed Scopus (1647) Google Scholar, 3Paridaens R. Dirix L. Lohrisch C. Beex L. Nooij M. Cameron D. Biganzoli L. Cufer T. Duchateau L. Hamilton A. Lobelle J.P. Piccart M. Ann. Oncol. 2003; 14: 1391-1398Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 4Baum M. Budzar A.U. Cuzick J. Forbes J. Houghton J.H. Klijn J.G. Sahmoud T. ATAC Trialists' GroupLancet. 2002; 359: 2131-2139Abstract Full Text Full Text PDF PubMed Scopus (1848) Google Scholar, 5Ellis M.J. Rosen E. Dressman H. Marks J. J. Steroid Biochem. Mol. Biol. 2003; 86: 301-307Crossref PubMed Scopus (34) Google Scholar). These compounds decrease aromatase activity indiscriminately at all body sites and thus cause severe estrogen deprivation. Recent evidence is indicative that local aromatase enzyme within the breast tumor tissue is the most critical source of estrogen for cancer cells (6Geisler J. J. Steroid Biochem. Mol. Biol. 2003; 86: 245-253Crossref PubMed Scopus (118) Google Scholar, 7Miller W.R. Dixon J.M. J. Steroid Biochem. Mol. Biol. 2001; 79: 93-102Crossref PubMed Scopus (82) Google Scholar, 8Miller W.R. Anderson T.J. Evans D.B. Krause A. Hampton G. Dixon J.M. J. Steroid Biochem. Mol. Biol. 2003; 86: 413-421Crossref PubMed Scopus (36) Google Scholar). Because the aromatase P450 (P450arom) 4The abbreviations used are: P450arom, aromatase P450; MCM, malignant breast epithelial cell-conditioned medium; CRE, cAMP response element; ATF-2, activating transcription factor-2; C/EBPβ, CCAAT/enhancer-binding protein-β; CBP, (cyclic AMP-response element-binding protein)-binding protein; Bt2cAMP, dibutyryl cyclic AMP; PDA, phorbol diacetate; BAF, breast adipose fibroblast; NaBu, sodium butyrate; DMEM, Dulbecco's modified Eagle's medium; ChIP, chromatin immunoprecipitation assay; FBS, fetal bovine serum; CREB, cAMP-response element-binding protein; RT, reverse transcriptase; EMSA, electrophoretic mobility shift assay; IP, immunoprecipitation. 4The abbreviations used are: P450arom, aromatase P450; MCM, malignant breast epithelial cell-conditioned medium; CRE, cAMP response element; ATF-2, activating transcription factor-2; C/EBPβ, CCAAT/enhancer-binding protein-β; CBP, (cyclic AMP-response element-binding protein)-binding protein; Bt2cAMP, dibutyryl cyclic AMP; PDA, phorbol diacetate; BAF, breast adipose fibroblast; NaBu, sodium butyrate; DMEM, Dulbecco's modified Eagle's medium; ChIP, chromatin immunoprecipitation assay; FBS, fetal bovine serum; CREB, cAMP-response element-binding protein; RT, reverse transcriptase; EMSA, electrophoretic mobility shift assay; IP, immunoprecipitation. gene is regulated by alternatively used distinct promoters in a signaling pathway-specific manner in multiple tissues, including the brain, bone, skin, and adipose tissue, it is possible to target the signaling pathway that regulates the P450arom gene primarily in breast cancer but to permit P450arom expression at other sites (9Simpson E. Clyne C. Speed C. Rubin G. Bulun S. Ann. N. Y. Acad. Sci. 2001; 949: 58-67Crossref PubMed Scopus (63) Google Scholar). This approach would potentially obviate total estrogen deprivation and may still be as effective as the currently used aromatase inhibitors.Aromatase P450 catalyzes the conversion of C19 steroids to estrogens in a number of human cells and tissues, e.g. ovarian granulosa cell and skin and adipose fibroblasts, placental syncytiotrophoblast, bone, and the brain (10McNatty K.P. Baird D.T. Bolton A. Chambers P. Corker C.S. MacLean H. J. Endocrinol. 1976; 71: 77-85Crossref PubMed Scopus (208) Google Scholar, 11Ackerman G.E. Smith M.E. Mendelson C.R. MacDonald P.C. Simpson E.R. J. Clin. Endocrinol. Metab. 1981; 53: 412-417Crossref PubMed Scopus (356) Google Scholar, 12Price T. Aitken J. Head J. Mahendroo M.S. Means G.D. Simpson E.R. J. Clin. Endocrinol. Metab. 1992; 74: 1247-1252Crossref PubMed Scopus (73) Google Scholar). P450arom expression in the adipose tissues is limited to undifferentiated fibroblasts and is not detected in significant quantities in the fully differentiated and lipid-filled adipocytes (10McNatty K.P. Baird D.T. Bolton A. Chambers P. Corker C.S. MacLean H. J. Endocrinol. 1976; 71: 77-85Crossref PubMed Scopus (208) Google Scholar, 11Ackerman G.E. Smith M.E. Mendelson C.R. MacDonald P.C. Simpson E.R. J. Clin. Endocrinol. Metab. 1981; 53: 412-417Crossref PubMed Scopus (356) Google Scholar, 12Price T. Aitken J. Head J. Mahendroo M.S. Means G.D. Simpson E.R. J. Clin. Endocrinol. Metab. 1992; 74: 1247-1252Crossref PubMed Scopus (73) Google Scholar). Aromatase activity in adipose fibroblasts has long been implicated in the pathophysiology of breast cancer growth (13Hemsell D.L. Grodin J.M. Brenner P.F. Siiteri P.K. MacDonald P.C. J. Clin. Endocrinol. Metab. 1974; 38: 476-479Crossref PubMed Scopus (389) Google Scholar, 14James V.H.T. Reed M.J. Lai L.C. Ghilchik M.W. Tait G.H. Newton C.J. Coldham N.G. Ann. N. Y. Acad. Sci. 1990; 595: 227-235Crossref PubMed Scopus (31) Google Scholar, 15O'Neill J.S. Elton R.A. Miller W.R. Br. Med. J. 1988; 296: 741-743Crossref PubMed Scopus (208) Google Scholar, 16Bulun S.E. Price T.M. Mahendroo M.S. Aitken J. Simpson E.R. J. Clin. Endocrinol. Metab. 1993; 77: 1622-1628Crossref PubMed Scopus (246) Google Scholar). Estrogen produced in breast adipose tissue acts locally to promote the growth of breast carcinomas (17Yue W. Wang J.P. Hamilton C.J. Demers L.M. Santen R.J. Cancer Res. 1998; 58: 927-932PubMed Google Scholar). Thus, the relationship between adipose stroma and breast cancer is unique in that the adipose fibroblasts provide structural and functional support for cancer growth. O'Neill et al. demonstrated that the breast quadrant displaying the highest level of aromatase activity was consistently involved with tumor (15O'Neill J.S. Elton R.A. Miller W.R. Br. Med. J. 1988; 296: 741-743Crossref PubMed Scopus (208) Google Scholar). Subsequently, we found that the highest levels of P450arom transcripts in adipose tissue from the quadrants bearing a tumor (12Price T. Aitken J. Head J. Mahendroo M.S. Means G.D. Simpson E.R. J. Clin. Endocrinol. Metab. 1992; 74: 1247-1252Crossref PubMed Scopus (73) Google Scholar). The clinical relevance of these observations has been exemplified by the fact that aromatase inhibitors are now the most commonly used noncytotoxic drugs in the treatment of breast cancer.Expression of P450arom is under the control of several distinct and partially tissue-specific promoters. The coding region of aromatase transcripts and thus the translated protein, however, are identical in each tissue site of expression (18Mahendroo M.S. Mendelson C.R. Simpson E.R. J. Biol. Chem. 1993; 268: 19463-19470Abstract Full Text PDF PubMed Google Scholar, 19Zhao Y. Mendelson C.R. Simpson E.R. Mol. Endocrinol. 1995; 9: 340-349Crossref PubMed Google Scholar) (Fig. 1). Three of these promoters (I.4, I.3, and II) are used in adipose tissue. In disease-free breast adipose tissue, P450arom is usually expressed at low levels via a distal promoter (I.4), whereas in the adipose tissue of the breast bearing a tumor, P450arom expression is increased through the activation of two proximal promoters, II and I.3, which are within the 0.7-kb region upstream to the common splice junction in the coding exon II (20Agarwal V.R. Bulun S.E. Leitch M. Rohrich R. Simpson E.R. J. Clin. Endocrinol. Metab. 1996; 81: 3843-3849Crossref PubMed Scopus (250) Google Scholar, 21Zhou C. Zhou D. Esteban J. Murai J. Siiteri P.K. Wilczynski S. Chen S. J. Steroid Biochem. Mol. Biol. 1996; 59: 163-171Crossref PubMed Scopus (127) Google Scholar, 22Harada N. J. Steroid Biochem. Mol. Biol. 1997; 61: 175-184Crossref PubMed Google Scholar) (Fig. 1). Because the activation of promoters I.3 and II is the critical molecular event responsible for the aberrant up-regulation of P450arom expression and thus local estrogen biosynthesis in breast tissues bearing a tumor, the possibility is presented then that promoter-specific inhibitors of P450arom could be developed to be used in the treatment of breast cancer.There may be multiple potential mechanisms responsible for the activation of promoters II and I.3 in human breast adipose fibroblasts (BAFs). Treatment of BAFs in serum-free medium with a cAMP analogue (e.g. Bt2cAMP) switches the promoter use to II and I.3 (18Mahendroo M.S. Mendelson C.R. Simpson E.R. J. Biol. Chem. 1993; 268: 19463-19470Abstract Full Text PDF PubMed Google Scholar, 19Zhao Y. Mendelson C.R. Simpson E.R. Mol. Endocrinol. 1995; 9: 340-349Crossref PubMed Google Scholar, 23Zhou D. Chen S. Arch. Biochem. Biophys. 1999; 371: 179-190Crossref PubMed Scopus (47) Google Scholar). The stimulation of promoters II/I.3 by cAMP is potentiated by PDA, a protein kinase C activator (18Mahendroo M.S. Mendelson C.R. Simpson E.R. J. Biol. Chem. 1993; 268: 19463-19470Abstract Full Text PDF PubMed Google Scholar). Simpson and co-workers (24Zhao Y. Agarwal V.R. Mendelson C.R. Simpson E.R. Endocrinology. 1996; 137: 5739-5742Crossref PubMed Scopus (364) Google Scholar) had also identified prostaglandin E2 as a potent factor that stimulates aromatase expression via promoters II/I.3. Prostaglandin E2 acts via E-prostanoid-2 (EP2) and EP1 receptor subtypes to stimulate both protein kinases A and C, respectively, and gives rise to strikingly high levels of promoter I.3- and II-specific P450arom transcripts (24Zhao Y. Agarwal V.R. Mendelson C.R. Simpson E.R. Endocrinology. 1996; 137: 5739-5742Crossref PubMed Scopus (364) Google Scholar). We and others had demonstrated that incubation of BAFs with malignant breast epithelial cell-conditioned medium induced aromatase expression via the activation of promoters I.3 and II (25Zhou J. Gurates B. Yang S. Sebastian S. Bulun S.E. Cancer Res. 2001; 61: 2328-2334PubMed Google Scholar, 26Harada N. Honda S. Breast Cancer Res. Treat. 1998; 49: S15-S21Crossref PubMed Google Scholar). Most recently, we demonstrated that malignant breast epithelial cell-conditioned medium (MCM) activated these promoters via a cAMP-independent pathway (25Zhou J. Gurates B. Yang S. Sebastian S. Bulun S.E. Cancer Res. 2001; 61: 2328-2334PubMed Google Scholar). The effects of tumor-conditioned medium appeared to be mediated by enhanced binding of transcription factor CCAAT/enhancer-binding protein (C/EBP)β to an NF-IL6 site (–317/–304) in the promoter I.3/II region (25Zhou J. Gurates B. Yang S. Sebastian S. Bulun S.E. Cancer Res. 2001; 61: 2328-2334PubMed Google Scholar).The factors secreted by malignant epithelial cells in MCM have not been identified yet (25Zhou J. Gurates B. Yang S. Sebastian S. Bulun S.E. Cancer Res. 2001; 61: 2328-2334PubMed Google Scholar). A mixture of factors in MCM seems to function in a redundant fashion to activate aromatase promoters I.3/II (25Zhou J. Gurates B. Yang S. Sebastian S. Bulun S.E. Cancer Res. 2001; 61: 2328-2334PubMed Google Scholar). We use MCM as a pathophysiologically relevant treatment, whereas Bt2cAMP + PDA is also employed as a surrogate hormonal treatment to mimic the downstream effects of MCM.Sodium butyrate (NaBu) is a four-carbon fatty acid, which is produced naturally in millimolar quantities during digestion by anaerobic bacteria in the cecum and colon (27McIntyre A. Young G.P. Taranto T. Gibson P.R. Ward P.B. Gastroenterology. 1991; 101: 1274-1281Abstract Full Text PDF PubMed Scopus (125) Google Scholar). NaBu exerts potent positive effects on growth arrest and cell differentiation and induces apoptosis in vitro in various malignant tumor cell lines, including breast cancer cell lines (28Weidle U.H. Grossmann A. Anticancer Res. 2000; 20: 1471-1485PubMed Google Scholar). Its therapeutic potential in experimental cancer models in mice had also been well documented, and some derivatives of NaBu had been clinically evaluated in a phase I study after oral administration in patients with solid tumors (28Weidle U.H. Grossmann A. Anticancer Res. 2000; 20: 1471-1485PubMed Google Scholar, 29Gore S.D. Carducci M.A. Exp. Opin. Investig. Drugs. 2000; 9: 2923-2934Crossref PubMed Scopus (114) Google Scholar). NaBu has been shown to inhibit histone deacetylase activity and induce histone hyperacetylation (30Sowa Y. Sakai T. Nippon Eiseigaku Zasshi. 2003; 58: 267-274Crossref PubMed Scopus (9) Google Scholar). NaBu is also a potent inducer of a serine-threonine phosphatase (31Cuisset L. Tichonicky L. Jaffray P. Delpech M. J. Biol. Chem. 1997; 272: 24148-24153Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The mechanism responsible for the anti-neoplastic activity of NaBu, however, has not been well understood to date.We used a model whereby NaBu was added to primary BAFs under various conditions to understand the molecular mechanism responsible for regulating aromatase expression from cancer-associated promoters II and I.3. We report here that the effects of NaBu on aromatase expression in breast tumor fibroblasts are mediated, at least in part, by a reduced phosphorylated state of ATF-2 at threonine 71 (Thr-71) and, consequently, disruption of a transcriptional complex containing phosphorylated ATF-2 (Thr-71), C/EBPβ, and CREB-binding protein (CBP) at the promoter II/I.3 regulatory region.EXPERIMENTAL PROCEDURESCell Cultures—Human adipose tissue samples were obtained at the time of surgery from women (n = 28) undergoing reduction mammoplasty following a protocol approved by the Institutional Review Board for Human Research of Northwestern University, Chicago. For primary human BAF cultures, adipose tissues were minced and digested with collagenase B (1 mg/ml) at 37 °C for 2 h. Single-cell suspensions were prepared by filtration through a 75-μm sieve. Fresh cells were suspended in DMEM/F-12 containing 10% FBS in a humidified atmosphere with 5% CO2 at 37 °C. Twelve to 24 h after the attachment of fibroblasts, culture medium was changed at 48-h intervals until the cells became confluent. At confluence, the cells were placed in serum-free medium for 24 h to wash out the effects of serum and then maintained in serum-free DMEM/F-12 conditioned by MCF-7 breast cancer cells (MCF-7-conditioned medium, MCM) or DMEM/F-12 containing various pharmacologic agents for varying intervals as depicted in relevant figures.We have employed human BAFs in primary culture since 1993 in this laboratory for studies related to regulation of aromatase expression (16Bulun S.E. Price T.M. Mahendroo M.S. Aitken J. Simpson E.R. J. Clin. Endocrinol. Metab. 1993; 77: 1622-1628Crossref PubMed Scopus (246) Google Scholar, 25Zhou J. Gurates B. Yang S. Sebastian S. Bulun S.E. Cancer Res. 2001; 61: 2328-2334PubMed Google Scholar, 32Nichols J.E. Bulun S.E. Simpson E.R. J. Soc. Gynecol. Investig. 1995; 2: 45-50Crossref PubMed Scopus (15) Google Scholar, 33Zhao Y. Nichols J.E. Bulun S.E. Mendelson C.R. Simpson E.R. J. Biol. Chem. 1995; 270: 16449-16457Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 34Meng L. Zhou J. Hironobu S. Suzuki T. Zeitoun K. Bulun S. Cancer Res. 2001; 61: 2250-2255PubMed Google Scholar). The cultured cells yield reproducible results for aromatase expression and do not demonstrate any significant subject-to-subject variation. Nevertheless, we repeated each experiment illustrated in Figs. 2, 3, 4, 5, 6, 7, 8, 9, 10 in cells from 3 to 6 different subjects and recorded reproducible results. Figs. 2, 3, 4, 5, 6, 7, 8, 9, 10 illustrate representative experiments. Cultures of T47D, Hep2G, and Jeg3 cell lines obtained from the ATCC and primary ovarian granulosa-lutein cells obtained during three in vitro fertilization cycles were performed as described previously (25Zhou J. Gurates B. Yang S. Sebastian S. Bulun S.E. Cancer Res. 2001; 61: 2328-2334PubMed Google Scholar, 35Gurates B. Amsterdam A. Tamura M. Yang S. Zhou J. Fang Z. Amin S. Sebastian S. Bulun S. Mol. Cell. Endocrinol. 2003; 208: 61-75Crossref PubMed Scopus (56) Google Scholar, 36Steinkampf M.P. Mendelson C.R. Simpson E.R. Mol. Endocrinol. 1987; 1: 465-471Crossref PubMed Scopus (121) Google Scholar).FIGURE 2NaBu inhibits P450arom expression via selectively silencing P450arom promoters I.3 and II in BAFs. A, PCR yields of promoter II- or I.3-specific P450arom transcripts showed linear increases in parallel to PCR amplification cycles using RNA from BAFs incubated in the presence or absence of MCM. B, both MCM and Bt2cAMP plus PDA stimulated P450arom transcript levels via activation of promoters II and I.3. The addition of NaBu strikingly decreased P450arom transcript levels via silencing of promoters II/I.3 but not I.4. The increase in I.4-specific transcript levels in response to NaBu did not give rise to a significant increase in total P450arom transcript levels as measured by amplification of the common coding region. After reaching confluence, BAFs were placed in serum-free medium for 24 h to remove the effects of serum. BAFs were then incubated under various conditions for a total of 48 h. Control, DMEM/F-12 only; NaBu, DMEM/F-12 + NaBu (15 mm); MCM, MCF-7 cell-conditioned medium; MCM + NaBu, MCM + NaBu (15 mm); Bt2cAMP + PDA, DMEM/F-12 + Bt2cAMP (0.5 mm) + PDA (100 nm); Bt2cAMP + PDA + NaBu, DMEM/F-12+ Bt2cAMP (0.5 mm) + PDA (100 nm) + NaBu (15 mm); no RT, no reverse transcriptase reaction mixture added as a negative control. NaBu was added 24 h prior to the treatment with MCM or Bt2cAMP plus PDA, and treatments with NaBu lasted for another 24 h together with other treatments (MCM or Bt2cAMP plus PDA) before harvesting the cells for RNA isolation. Total RNA was subjected to exon-specific RT-PCR to amplify promoter-specific untranslated first exons or the common coding region (for total P450arom transcript levels). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified to control the integrity of RNA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Time kinetics and dose response for the inhibitory effects of NaBu. BAFs were incubated in MCM for 24 h. We then added 15 mm NaBu and continued incubation for various periods, or we maintained these cells in MCM containing various concentrations of NaBu for another 24 h. Total RNA was isolated and subjected to exon-specific RT-PCR to amplify promoter-specific untranslated first exons of P450arom transcripts. A, the time kinetics of inhibitory effects of NaBu on promoters II and I.3. B, NaBu decreased promoter II- and I.3-specific transcripts in a dose-dependent manner. C, staurosporine (10 nm) or cycloheximide (30 μm) reversed the inhibitory effect of NaBu, whereas neither SQ22,536 (100 μm) nor sodium orthovanadate (5 μm) altered the NaBu effect. As expected, actinomycin D (5 μg/ml) decreased MCM-stimulated mRNA levels. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4Regulation of aromatase activity and promoter usage by various hormones or NaBu in four distinct human cell types that use alternative promoters for aromatase expression. Cells were maintained in serum-free DMEM/F-12 medium for 12 h and then incubated with the labeled substrate androstenedione under various conditions. Control, DMEM/F-12; LET, letrozole 10–8 m; cAMP + PDA, Bt2cAMP (0.5 mm) + PDA (100 nm) in DMEM/F-12; MCM, MCF-7 cell-conditioned DMEM/F-12; DEX + ser, dexamethasone 5 × 10–7 m and 10% fetal bovine serum in DMEM/F-12; NaBu, NaBu (15 mm). Assay conditions are described under “Experimental Procedures.” Enzyme activity in the absence of a treatment is taken as 100%. The error bars represent means ± S.E. for each treatment that was carried out in triplicate replicates. Primary promoter usage was determined by untranslated first exon-specific RT-PCR applied to total RNA isolated from cells incubated under identical conditions. Aromatase activity in each cell type treated with the aromatase enzyme inhibitor letrozole (LET) was undetectable. Statistically significant differences between treatment pairs were indicated as follows. A, *, p < 0.001; **, p < 0.001. B,*, p < 0.01; **, p < 0.01; #, p < 0.05. C and D, NaBu did not regulate aromatase activity significantly in HepG2 or JEG3 cell lines, in which promoters I.3/II are not important regulators of the P450arom gene.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5Regulation of the P450arom promoter I.3/II region by Bt2cAMP + PDA, MCM, and NaBu in BAFs. Luciferase plasmids containing the –517/–16 bp-5′-flanking region containing P450arom promoters II and I.3 were transfected into BAFs. pCMV Renilla was used as an internal control for transfection efficiency. Promoter II/I.3 activity was normalized to pCMV Renilla and was represented as the average of data from triplicate replicates ± S.E. The empty luciferase vector PGL3-basic was arbitrarily assigned a unit of 1, and the rest of the results were expressed as multiples of the PGL3-basic vector. A, statistically significant differences were observed between the following treatment pairs: *, p < 0.01; **, p < 0.001; #, p < 0.001. B, selective mutations of the NF-IL6 site at –317/–304 or CRE at –211/–199 significantly decreased basal promoter activity and induction by MCM. #, p < 0.01; †, p < 0.001; *, p < 0.005; **, p < 0.005.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6MCM enhances and NaBu reduces recruitment of phosphorylated ATF-2 by a CRE (–211/–199) at promoter I.3/II region. EMSA was performed using an oligonucleotide probe (–214/–192 bp) containing the –211/–199-bp CRE, 1 μg of nuclear extract (NE) from BAFs incubated with MCM or MCM plus NaBu (15 mm), and antibodies against nonphospho-specific ATF-2 or phosphorylated ATF-2 at Thr-71 (pATF-2). We identified a specific complex as verified by a cold competitor probe. The complex had a similar size in BAFs treated with MCM in the presence or absence of NaBu. Antibodies against ATF-2 (nonphospho-specific) or pATF-2 depleted the complex in BAFs treated with MCM, indicating the presence of nonphosphorylated and phosphorylated ATF-2 in this complex. On the other hand, the complex in BAFs treated with MCM plus NaBu was depleted only by the anti-ATF-2 antibody (nonphospho-specific) indicating the absence of phosphorylated ATF-2 in the complex.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 7Binding of phosphorylated ATF-2 (Thr-71), C/EBPβ, and CBP to promoter I.3/II regulatory region in BAFs is enhanced by MCM and abolished by NaBu. Findings depicted in Fig. 6 suggested that MCM increased and NaBu decreased the phosphorylated state of ATF-2 that occupies a CRE in the promoter I.3/II region. We confirm these observations by ChIP, and we also demonstrate that pATF-2 likely stabilizes a complex composed of pATF-2, C/EBPβ, and CBP at this promoter region. BAFs were maintained in serum-free DMEM/F-12 for 24 h and then incubated under various conditions for a total of 36 h (no treatment, DMEM/F-12 only; NaBu, DMEM/F-12 + NaBu (15 mm); MCM, MCF-7 cell-conditioned medium; MCM + NaBu, MCM + NaBu (15 mm)). Treatments lasted for 12 h. For the treatment with MCM plus NaBu, NaBu was added 24 h prior to the addition of MCM, and incubation in MCM plus NaBu lasted for another 12 h. Cells were cross-linked with 1% formaldehyde followed by immunoprecipitation using various antibodies. A, anti-histone H3 and normal rabbit IgG were used as positive and negative controls for the ChIP assay, respectively. We amplified by PCR the –511/–194-bp sequence in the P450arom promoter II/I.3 regulatory region (see Fig. 1). The intensities of PCR fragments correlate with the in situ binding affinities of these factors to the –511/–194-bp region. Both MCM and NaBu increased the quantities of acetylated histones, which were detected by both anti-acetylated histone H3/H4 and anti-acetylated lysine antibodies. In contrast to acetylated histone H3/H4, binding affinities of phosphorylated ATF-2 (Thr-71), C/EBPβ, and CBP to the promoter II/I.3 regulatory region were specifically enhanced by MCM and inhibited by NaBu. On the other hand, binding affinities of factors that could be immunoprecipitated by a nonphospho-specific ATF-2 was not altered by these treatments. B, staurosporine (10 nm) reversed NaBu-dependent inhibition of pATF-2 binding to the P450arom promoter, whereas calyculin-A (80 nm) did not alter" @default.
• W2100392901 created "2016-06-24" @default.
• W2100392901 creator A5005820169 @default.
• W2100392901 creator A5009298625 @default.
• W2100392901 creator A5024255678 @default.
• W2100392901 creator A5025106342 @default.
• W2100392901 creator A5025575486 @default.
• W2100392901 creator A5083128911 @default.
• W2100392901 creator A5091247413 @default.
• W2100392901 date "2006-02-01" @default.
• W2100392901 modified "2023-09-27" @default.
• W2100392901 title "A Novel Role of Sodium Butyrate in the Regulation of Cancer-associated Aromatase Promoters I.3 and II by Disrupting a Transcriptional Complex in Breast Adipose Fibroblasts" @default.
• W2100392901 cites W1453600927 @default.
• W2100392901 cites W1495237614 @default.
• W2100392901 cites W1547162940 @default.
• W2100392901 cites W1579473562 @default.
• W2100392901 cites W1897037552 @default.
• W2100392901 cites W1933411091 @default.
• W2100392901 cites W1965722126 @default.
• W2100392901 cites W1973241341 @default.
• W2100392901 cites W1975064171 @default.
• W2100392901 cites W1977137328 @default.
• W2100392901 cites W1999582107 @default.
• W2100392901 cites W2002196249 @default.
• W2100392901 cites W2006398865 @default.
• W2100392901 cites W2013376255 @default.
• W2100392901 cites W2021031249 @default.
• W2100392901 cites W2021800802 @default.
• W2100392901 cites W2022380310 @default.
• W2100392901 cites W2022713347 @default.
• W2100392901 cites W2024178675 @default.
• W2100392901 cites W2025393772 @default.
• W2100392901 cites W2026264021 @default.
• W2100392901 cites W2026898742 @default.
• W2100392901 cites W2027928836 @default.
• W2100392901 cites W2029365043 @default.
• W2100392901 cites W2029725226 @default.
• W2100392901 cites W2033880121 @default.
• W2100392901 cites W2047786508 @default.
• W2100392901 cites W2052115456 @default.
• W2100392901 cites W2073030463 @default.
• W2100392901 cites W2076823272 @default.
• W2100392901 cites W2078297169 @default.
• W2100392901 cites W2079681040 @default.
• W2100392901 cites W2082717337 @default.
• W2100392901 cites W2083329762 @default.
• W2100392901 cites W2086235375 @default.
• W2100392901 cites W2090793581 @default.
• W2100392901 cites W2091969987 @default.
• W2100392901 cites W2092171701 @default.
• W2100392901 cites W2101881391 @default.
• W2100392901 cites W2108477359 @default.
• W2100392901 cites W2109525253 @default.
• W2100392901 cites W2122095366 @default.
• W2100392901 cites W2126100083 @default.
• W2100392901 cites W2133853086 @default.
• W2100392901 cites W2137385309 @default.
• W2100392901 cites W2153767846 @default.
• W2100392901 cites W2163688875 @default.
• W2100392901 cites W2166434116 @default.
• W2100392901 cites W4248479347 @default.
• W2100392901 doi "https://doi.org/10.1074/jbc.m508498200" @default.
• W2100392901 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16303757" @default.
• W2100392901 hasPublicationYear "2006" @default.
• W2100392901 type Work @default.
• W2100392901 sameAs 2100392901 @default.
• W2100392901 citedByCount "42" @default.
• W2100392901 countsByYear W21003929012012 @default.
• W2100392901 countsByYear W21003929012014 @default.
• W2100392901 countsByYear W21003929012015 @default.
• W2100392901 countsByYear W21003929012016 @default.
• W2100392901 countsByYear W21003929012017 @default.
• W2100392901 countsByYear W21003929012018 @default.
• W2100392901 countsByYear W21003929012019 @default.
• W2100392901 countsByYear W21003929012020 @default.
• W2100392901 countsByYear W21003929012021 @default.
• W2100392901 countsByYear W21003929012022 @default.
• W2100392901 crossrefType "journal-article" @default.
• W2100392901 hasAuthorship W2100392901A5005820169 @default.
• W2100392901 hasAuthorship W2100392901A5009298625 @default.
• W2100392901 hasAuthorship W2100392901A5024255678 @default.
• W2100392901 hasAuthorship W2100392901A5025106342 @default.
• W2100392901 hasAuthorship W2100392901A5025575486 @default.
• W2100392901 hasAuthorship W2100392901A5083128911 @default.
• W2100392901 hasAuthorship W2100392901A5091247413 @default.
• W2100392901 hasBestOaLocation W21003929011 @default.
• W2100392901 hasConcept C101762097 @default.
• W2100392901 hasConcept C104317684 @default.
• W2100392901 hasConcept C121608353 @default.
• W2100392901 hasConcept C126322002 @default.
• W2100392901 hasConcept C134018914 @default.
• W2100392901 hasConcept C150194340 @default.
• W2100392901 hasConcept C171089720 @default.
• W2100392901 hasConcept C185592680 @default.
• W2100392901 hasConcept C2776166826 @default.
• W2100392901 hasConcept C2778942008 @default.
• W2100392901 hasConcept C502942594 @default.
• W2100392901 hasConcept C530470458 @default.

• next