Identification and Characterization of a Novel Factor That Regulates Quinone Reductase Gene Transcriptional Activity
2000; Elsevier BV; Volume: 275; Issue: 44 Linguagem: Inglês
10.1074/jbc.m003880200
ISSN1083-351X
AutoresMonica M. Montano, Bryan M. Wittmann, Nicole R. Bianco,
Tópico(s)Bioactive Compounds and Antitumor Agents
ResumoThe regulation of the quinone reductase (QR) gene as well as other genes involved in detoxification is known to be mediated by an electrophile/antioxidant response element (EpRE/ARE). We have previously observed that QR is up-regulated by the antiestrogen trans-hydroxytamoxifen in breast cancer cells. QR gene regulation by the antiestrogen-occupied estrogen receptor (ER) is mediated by the EpRE-containing region of the human QR gene, and the ER is one of the complex of proteins that binds to the EpRE. In an effort to further understand the mechanism for ER regulation of QR gene we identified other protein factors that regulate QR gene transcriptional activity in breast cancer cells. One of these protein factors, hPMC2 (human homolog of Xenopus gene whichprevents mitotic catastrophe), directly binds to the EpRE and interacts with the ER in yeast genetic screening and in vitro assays. Interestingly hPMC2 interacts more strongly to ERβ when compared with ERα. In transient transfection assays using reporter constructs containing the EpRE, hPMC2 alone can slightly activate reporter in ER-negative MDA-MB-231 breast cancer cells. The activation of QR gene activity by hPMC2 is enhanced in the presence of ERβ. The regulation of the quinone reductase (QR) gene as well as other genes involved in detoxification is known to be mediated by an electrophile/antioxidant response element (EpRE/ARE). We have previously observed that QR is up-regulated by the antiestrogen trans-hydroxytamoxifen in breast cancer cells. QR gene regulation by the antiestrogen-occupied estrogen receptor (ER) is mediated by the EpRE-containing region of the human QR gene, and the ER is one of the complex of proteins that binds to the EpRE. In an effort to further understand the mechanism for ER regulation of QR gene we identified other protein factors that regulate QR gene transcriptional activity in breast cancer cells. One of these protein factors, hPMC2 (human homolog of Xenopus gene whichprevents mitotic catastrophe), directly binds to the EpRE and interacts with the ER in yeast genetic screening and in vitro assays. Interestingly hPMC2 interacts more strongly to ERβ when compared with ERα. In transient transfection assays using reporter constructs containing the EpRE, hPMC2 alone can slightly activate reporter in ER-negative MDA-MB-231 breast cancer cells. The activation of QR gene activity by hPMC2 is enhanced in the presence of ERβ. quinone reductase estrogen receptor electrophile response element trans-hydroxytamoxifen antioxidant response element estrogen receptor glutathioneS-transferases polymerase chain reaction green fluorescent protein estrogen receptor response element 12-O-tetradecanoylphorbol-13-acetate response element Phase 2 detoxification enzymes such as NAD(P)H:(quinone-acceptor) oxidoreductase (quinone reductase (QR)),1 glutathioneS-transferases (GSTs), epoxide hydrolase, and UDP-glucuronosyltransferases are induced in cells by electrophilic compounds and phenolic antioxidants (reviewed in Refs. 1Talalay P. Adv. Enzyme Regul. 1989; 28: 237-250Crossref PubMed Scopus (195) Google Scholar and 2Prestera T. Zhang Y. Spencer S.R. Wilczak C.A. Talalay P. Adv. Enzyme Regul. 1993; 33: 281-296Crossref PubMed Scopus (257) Google Scholar). These widely distributed enzymes detoxify electrophiles, thereby protecting cells against the toxic and neoplastic effects of carcinogens. We have previously shown that increases in QR enzyme activity can be induced by low concentrations of antiestrogens in breast cancer cells (3Montano M.M. Katzenellenbogen B.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2581-2586Crossref PubMed Scopus (150) Google Scholar). Induction of QR enzymatic activity showed unusual reversed pharmacology, being markedly up-regulated by antiestrogen and suppressed by estrogen in breast cancer cells. The antiestrogen regulation of quinone reductase enzymatic activity represents a potentially important pharmacological mechanism for this group of anticancer drugs that had not been previously recognized. The electrophile response element (EpRE) motif has been identified in the regulatory region of the genes encoding QR and the GST-Ya subunit (4Favreau L.V. Pickett C.B. J. Biol. Chem. 1991; 266: 4556-4561Abstract Full Text PDF PubMed Google Scholar, 5Rushmore T.H. King R.G. Paulson K.E. Pickett C.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3826-3830Crossref PubMed Scopus (385) Google Scholar). This element has been shown to mediate basal expression and its activation by phenolic antioxidants (6Friling R.S. Bensimon A. Tichauer Y. Daniel V. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6258-6262Crossref PubMed Scopus (422) Google Scholar, 7Rushmore T.H. Morton M.R. Pickett C.B. J. Biol. Chem. 1991; 266: 11632-11639Abstract Full Text PDF PubMed Google Scholar, 8Prestera T. Talalay P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8965-8969Crossref PubMed Scopus (218) Google Scholar, 9Xie T. Belinsky M. Xu Y. Jaiswal A.K. J. Biol. Chem. 1995; 270: 6894-6900Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 10Jaiswal A.K. Biochemistry. 1991; 30: 10647-10653Crossref PubMed Scopus (195) Google Scholar), and it appears to be essential for antiestrogen stimulation (3Montano M.M. Katzenellenbogen B.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2581-2586Crossref PubMed Scopus (150) Google Scholar). QR expression is up-regulated at the transcriptional level through the EpRE by antiestrogen-liganded estrogen receptor (ER). Interestingly ERβ is a more potent activator of QR gene transcriptional activity than ERα. Gel shift assays suggest that antiestrogen-mediated induction of QR gene transcriptional activity in MCF7 cells involves a direct transcriptional effect where ERα or ERβ are components of the protein complex that binds the EpRE. However several aspects of antiestrogen regulation of QR transcriptional activity cannot be attributed solely to ER binding to the EpRE and remain to be investigated. This is especially true in light of our previous observations that 1) the time course of induction of QR enzyme activity is relatively slow (with increases in QR mRNA first detectable at 12–16 h after antiestrogen treatment of MCF7 cells (3Montano M.M. Katzenellenbogen B.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2581-2586Crossref PubMed Scopus (150) Google Scholar)), 2) antiestrogen activation of GST Ya gene transcriptional activity is mediated through an EpRE that is not homologous to the ERE (3Montano M.M. Katzenellenbogen B.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2581-2586Crossref PubMed Scopus (150) Google Scholar), and 3) the interaction of the ER with the EpRE is weak, and the EpRE interacts with additional proteins (12Montano M.M. Jaiswal A.K. Katzenellenbogen B.S. J. Biol. Chem. 1999; 273: 25443-25449Abstract Full Text Full Text PDF Scopus (107) Google Scholar). Clearly the regulation by antiestrogen-liganded ER may be also attributable to changes in the levels and/or the activity of other factors. Thus studies, now reported here, were conducted to further dissect the molecular mechanism(s) involved in antiestrogen induction of QR activity and identify other transcriptional factors involved in this regulation. We used yeast genetic screenings to identify protein factors that fulfill two criteria, the ability to bind to the EpRE and interact with the ER. We report the identification and characterization of hPMC2 as a factor that fulfills these two criteria in yeast genetic screenings as well as in vitro assays. Although only a slight up-regulation of QR gene transcriptional activity was observed with hPMC2, antiestrogen-liganded ERβ enhanced hPMC2-mediated activation of QR transcriptional activity. Cell culture media were purchased from Life Technologies, Inc. Calf serum was from Hyclone Laboratories (Logan, UT), and fetal calf serum was from Sigma. The antiestrogen trans-hydroxytamoxifen (TOT) was obtained from Sigma. tert-Butylhydroquinone was obtained from Aldrich. Custom oligonucleotides were purchased from Genosys (Grand Island, NY). The reporter constructs for one-hybrid screenings were constructed by cloning one copy of the EpRE into the pHISi-1 and pLacZi vector (CLONTECH, Palo Alto, CA) to make EpRE-pHISi-1 and EpRE-pLacZi. The following oligonucleotides, which contain the −476 to −437 region of the human QR gene, were used: EpRE-1, 5′-AATTAAATCGCAGTCACAGTGACTCAGCAGAATCTGAGCCTAGG -3′; EpRE-2, 5′-TCGACCTAGGCTCAGATTCTGCTGAGTCACTGTGACTGCGATTT-3′; EpRE-3, 5′-CGCGCCTAGGCTCAGATTCTGGTGAGTCACTGTGACTGCGATTT-3′. EpRE 1 and 2 were annealed, gel-purified, and cloned intoEcoRI/SalI-digested pLacZi vector. EpRE 1 and 3 were annealed, gel-purified, and cloned intoEcoRI/MluI-digested pHISi-1. The oligonucleotides containing mutated EpRE, 5′-AATTAAATCGCAGTCACAGGTCAGACGCAGAATCTGAGCCTAGG-3′ and 5′-TCGACCTAGGCTCAGATTCTGCGTCTGACCTGTGACTGCGATTT-3′, were annealed, gel-purified, and cloned intoEcoRI/SalI-digested pLacZi vector to make EpREmut-pLacZi. pNQO1CAT 0.863 (containing 863 base pairs of the QR gene promoter, which contains one copy of the EpRE between −476 and −437), pNQO1 CAT 0.365 (containing 365 base pairs of the QR gene promoter and missing the EpRE), pNQO1hARE-tk-CAT (containing the region between −476 and −446 of the QR gene promoter introduced upstream of the thymidine kinase gene promoter in the pBLCAT2 vector), pNQO1hARE(mut)-tk-CAT (containing a mutated TRE element), and pNQO1hARE(mut2)-tk-CAT (containing a mutated TRE-like element) have been described previously (9Xie T. Belinsky M. Xu Y. Jaiswal A.K. J. Biol. Chem. 1995; 270: 6894-6900Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 10Jaiswal A.K. Biochemistry. 1991; 30: 10647-10653Crossref PubMed Scopus (195) Google Scholar, 12Montano M.M. Jaiswal A.K. Katzenellenbogen B.S. J. Biol. Chem. 1999; 273: 25443-25449Abstract Full Text Full Text PDF Scopus (107) Google Scholar). The partial hPMC2 clone, pAD-GAL4–2.1-hPMC2(139–422), obtained from yeast two-hybrid screening contains amino acids 139–422 of human homolog of XPMC2 cloned in-frame with the activation domain of GAL4 in the pAD-GAL4–2.1 phagemid vector (Stratagene, La Jolla, CA). The partial cDNA clone was released by BamHI/SalI digestion and inserted in-frame with the FLAG epitope intoBamHI/SalI-digested pCMV-Tag2B vector (Stratagene) to make pCMV-Tag2B-hPMC2(139–422). cDNA encoding amino acids 1–204 was obtained using Access reverse transcriptase-PCR kit from Promega (Madison, WI). Briefly, DNA-free RNA was obtained by treatment of total RNA with DNaseI in the presence of placental RNase inhibitor for 30 min at 37 °C. After phenol/chloroform extraction and ethanol precipitation, reverse transcriptase-PCR reactions were performed for each RNA sample using 0.25 μg of DNA-free total RNA in 1× reaction buffer, 0.2 mm each of dATP, dCTP, dGTP, and dTTP, 1 mm MgSO4, 5 units of avian myeloblastosis virus reverse transcriptase, 5 units of Tfl DNA polymerase, and 1 μm each of upstream primer (CGGCCCAGGCGCTGGACGGCAG) and downstream primer (CTATGGCAGCTTCGATATCCGGTG). The first strand cDNA synthesis reactions were performed at 48 C for 45 min, 94 C for 2 min. The second strand cDNA synthesis and PCR amplification reactions were performed at 40 cycles of 94 °C for 30 s, 60 °C for 1 min, 68 °C for 2 min, followed by a final extension step at 68 °C for 7 min. Amplified cDNA was run on a 1.2% agarose gel and purified using the QIAEX kit from Qiagen (Chatsworth, CA). The cDNA was then cloned into the pCR-Blunt II-TOPO vector to make pCRII-TOPO-hPMC2(1–204) using the Zero Blunt TOPO PCR cloning kit from Invitrogen (Carlsbad, CA) and sequenced using the Sequenase Kit (U. S. Biochemical Corp.). To construct a mammalian expression vector for full-length hPMC2 cDNA, pCRII-TOPO-hPMC2(1–204) was digested with BamHI and EcoRV to release the fragment encoding amino acids 1–204. The fragment was then inserted in-frame with the FLAG epitope at the 5′ end and in-frame with amino acids 205–422 at the 3′ end intoBamHI/EcoRV-digested pCMV-Tag2B-hPMC2(139–422) to make pCMV-Tag2B-hPMC2. pGEX2T-hPMC2, which encodes full-length and FLAG-epitope tagged hPMC2 in-frame with GST was constructed byNotI/XhoI digestion of pCMV-Tag2B-hPMC2. The insert, which contains cDNA encoding full-length FLAG-hPMC2, was blunted with Klenow and inserted into BamHI-digested and -blunted pGEX2T (Amersham Pharmacia Biotech). pEGFP-hPMC2, which encodes full-length hPMC2 in-frame with the coding sequence for green fluorescent protein (GFP), was constructed byBamHI/XhoI digestion of pCMV-Tag2B-hPMC2. The insert was blunted and cloned into BglII-digested and -blunted pEGFP-C3 vector (CLONTECH). The expression vectors for the wild type human estrogen receptor α and β have been described previously (12Montano M.M. Jaiswal A.K. Katzenellenbogen B.S. J. Biol. Chem. 1999; 273: 25443-25449Abstract Full Text Full Text PDF Scopus (107) Google Scholar, 13Wrenn C.K. Katzenellenbogen B.S. J. Biol. Chem. 1993; 268: 24089-24098Abstract Full Text PDF PubMed Google Scholar). To construct pBD-GAL4-ERβ(EF), which encodes the EF domain of ERβ cloned in-frame with the DNA binding domain of GAL4, CMV-ERβ was digested with KpnI/SmaI, blunted, and inserted intoSalI-digested, blunted pBD-GAL4-Cam (Stratagene). To construct pGEX2T-ERβ, which encodes full-length and FLAG epitope-tagged ERβ in-frame with GST, FLAG-ERβ-BSII-SK+ was digested with XbaI/HindIII. The insert was blunted and cloned into BamHI-digested and -blunted pGEX2T. The plasmid pCMVβ (CLONTECH), which encodes the β-galactosidase gene, was used as an internal control for transfection efficiency in all experiments. To identify putative EpRE-interacting proteins, the EpRE-containing reporter vectors, EpRE-pHISi-1 and EpRE-pLacZi (CLONTECH), were integrated into the yeast strain YM4271 genome sequentially. As a negative control the mutated EpRE-containing reporter vector, EpREmut-pLacZi, was introduced separately into the YM4271 yeast strain. Yeast cells containing the EpRE-pHISi-1 and EpRE-pLacZi reporter vectors were cotransformed with a human MCF7 (a breast cancer epithelial cell line) cDNA library in pAD-GAL4 (14Montano M.M. Ekena K. Delage-Mourroux R. Chang W. Martini P. Katzenellenbogen B.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6947-6952Crossref PubMed Scopus (242) Google Scholar) and plated on media lacking histidine and supplemented with 50 mm3-aminotriazole. 3-Aminotriazole is a competitive inhibitor of the yeast HIS3 protein, which suppresses the basal level of expression ofHIS3. β-Galactosidase activities were determined fromHIS3 + colonies using both filter lift or liquid assays (14Montano M.M. Ekena K. Delage-Mourroux R. Chang W. Martini P. Katzenellenbogen B.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6947-6952Crossref PubMed Scopus (242) Google Scholar). HIS3 + colonies exhibiting high β-galactosidase activity (LacZ + colonies) were further tested for interaction with the estrogen receptor. The yeast two hybrid screenings used to test interactions of putative EpRE-interacting clones with the estrogen receptor were described previously (14Montano M.M. Ekena K. Delage-Mourroux R. Chang W. Martini P. Katzenellenbogen B.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6947-6952Crossref PubMed Scopus (242) Google Scholar). To establish interaction of promising clones with ERβ, a standard two-hybrid strategy was used wherein library plasmids encoding putative EpRE-interacting clones were also transformed into YRG2 yeast strain containing pBD-GAL-ERβ(EF) and plated into media lacking histidine. β-Galactosidase activities were determined fromHIS3 + colonies. To recover library plasmids, total DNA from HIS3 + ,LacZ + colonies was isolated and used to transformEscherichia coli (XLI-Blu MRF′ strain from Stratagene). To ensure that the correct cDNAs were isolated, library plasmids were transformed back into the YM4271 yeast strain containing EpRE-pHISi-1 and EpRE-pLacZi, or EpREmut-pLacZi reporter vectors, plated onto media lacking histidine, and tested for β-galactosidase activity. The cDNAs were also transformed back into the YRG2 yeast strain containing pBD-GAL-ERβ(EF). Crude extracts were obtained from E. coli transformed with pGEX2T-hPMC2 or pGEX2T-ERβ and induced with isopropyl-β-d-thiogalactopyranoside (Promega). Five hundred micrograms of crude extract containing GST fusion proteins were incubated overnight at 4 °C with 50 μl of glutathione-Sepharose beads (50% slurry; Amersham Pharmacia Biotech). After three washes with 1 ml of NET (150 mm NaCl, 5 mm EDTA, 50 mm Tris, pH 7.4), the beads were incubated overnight at 4 °C with 25 μl (or 1.25 units) of thrombin protease in 1× PBS. After protease treatment, the beads were spun down, and the supernatants, which contain purified hPMC2 or ERβ, were collected. Aliquots of the protein samples were analyzed by SDS-polyacrylamide gel electrophoresis protein assay kit (Pierce) before being utilized in gel shift assays to check protein quality and relative purity and total protein concentration in extracts, respectively. As a negative control, crude extracts fromE. coli transformed with pGEX2T (i.e. lacking hPMC2 or ERβ coding sequence) were subjected to the same purification and protease treatment procedure. The single-stranded oligomers, either 5′-AAT TAA ATC GCA GTC ACA GTG ACT CAG CAG AAT CTG AGC CTA GG -3′, which contains the −476 to −437 region of the human QR gene or 5′-AGC TAG TCA GGT CAC AGT GAC CTG ATC-3′, which contains the consensus ERE, were annealed to their complement. The resultant double-stranded oligomer was gel-purified on a nondenaturing 4.5% polyacrylamide gel run in 0.5× Tris-buffered EDTA. The ability of purified protein(s) to bind to the EpRE was analyzed using gel mobility shift assays as described previously (12Montano M.M. Jaiswal A.K. Katzenellenbogen B.S. J. Biol. Chem. 1999; 273: 25443-25449Abstract Full Text Full Text PDF Scopus (107) Google Scholar). Briefly, 4 μl (10–100 ng) of purified proteins were mixed with 1 ng of end-labeled EpRE oligomer in the presence of 0.4 μg/μl dI-dC, 20 mm HEPES, 200 mm KCl, 10 mm MgCl2, 2 mm dithiothreitol, 2 mm EDTA, 20% glycerol, 1 μg/μl bovine serum albumin and incubated at room temperature for 20 min. The specificity of binding was assessed by competition with excess unlabeled double-stranded EpRE, mutated EpRE, or ERE. The non-denaturing gels used to analyze the protein-DNA complexes were run as described previously (15Kraus W.L. Montano M.M. Katzenellenbogen B.S. Mol. Endocrinol. 1994; 8: 952-969Crossref PubMed Scopus (176) Google Scholar). The FLAG antibody M2 was obtained from Sigma. The ERβ polyclonal antibody was obtained from the laboratory of Benita S. Katzenellenbogen (University of Illinois, Champaign-Urbana, IL). In vitrotranscription and translation of ERα and ERβ were performed using the Promega TNT kit. Briefly, 1 μg of ERα-BSII-SK+ or ERβ-BSII-SK+ (12Montano M.M. Jaiswal A.K. Katzenellenbogen B.S. J. Biol. Chem. 1999; 273: 25443-25449Abstract Full Text Full Text PDF Scopus (107) Google Scholar) were mixed with 25 μl of TNT rabbit reticulocyte lysate, 2 μl of TNT buffer, 1 μl of amino acid mixture, 1 μl of T3 RNA polymerase (20 U/μl), and 4 μl of [35S]methionine (15 μCi/μl; ICN, Costa Mesa, CA). The final reaction of 50 μl was incubated for 90 min at 30 °C. For the in vitrointeraction assays, 500 μg of E. coli bacteria crude extracts containing GST-hPMC2 fusion protein were incubated at 4 °C with 50 μl of glutathione-Sepharose beads (50% slurry; Amersham Pharmacia Biotech) for 2.5 h. After two washes with 1 ml of NENT (100 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40, 20 mm Tris, pH 7.9, 0.5% milk) and two washes with 1 ml of binding buffer (20 mm HEPES, pH 7.9, 10% v/v glycerol, 60 mm NaCl, 1 mm dithiothreitol, 6 mmMgCl2, 1 mm EDTA), the beads were incubated with 5 μl of in vitro translated ERα or ERβ overnight at 4 °C. The beads were then washed 3 times with 1 ml of NET and 2 times with 1 ml of binding buffer. After washing, bound protein was eluted with 10 mm reduced glutathione in 50 mmTris-HCl, pH 8.0, and boiled in SDS sample buffer. The protein samples were then analyzed by SDS-polyacrylamide gel electrophoresis. The gel was dried, and radiolabeled protein was detected by autoradiography. MDA-MB-231 and HepG2 cells were maintained and transfected as described previously (3Montano M.M. Katzenellenbogen B.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2581-2586Crossref PubMed Scopus (150) Google Scholar, 16Chusacultanachai S. Glenn K.A. Rodriguez A.O. Read E.K. Gardner J.F. Katzenellenbogen B.S. Shapiro D.J. J. Biol. Chem. 1999; 274: 23591-23598Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Cells were seeded for transfection in a 100-mm dish in improved minimum essential media minus phenol red containing 5% charcoal dextran treated calf serum. Cells at 30–50% confluence were transfected by the CaPO4 coprecipitation method 48 h later with 2 μg of EpRE (wild type or mutant)-containing reporter constructs, ERβ expression vector, hPMC2 expression vector, and 1.5 μg of pCMVβ β-galactosidase internal control plasmid. Carrier DNA pTZ19R was added to adjust the total DNA to 15 μg. Cells remained in contact with the precipitate for 5 h and were then subjected to a 2.5-min glycerol shock (20% glycerol in improved minimum essential media minus phenol red plus 5% charcoal dextran treated calf serum). Cells were rinsed with Hepes-buffered saline solution and given fresh media with or without hormones. All cells were harvested 24 h after hormone treatment. Extracts were prepared in 200 μl of 250 mm Tris HCl, pH 7.5, using three freeze-thaw cycles. β-Galactosidase activity, which was measured to normalize for transfection efficiency, and CAT activity were assayed as described previously (17Reese J.C. Katzenellenbogen B.S. J. Biol. Chem. 1991; 266: 10880-10887Abstract Full Text PDF PubMed Google Scholar). The pEGFP-hPMC2 vector was transfected into MDA-MB-231 cells as described above. As a control, separate plates of cells were transfected with pEGFP-C3 vector alone. Twenty-four hours after glycerol shock, the cells were observed by both phase contrast and fluorescence microscopy. Data were evaluated by analysis of variance and tested for statistical significance using Student'st test. Yeast genetic screenings were used to identify protein factors expressed in breast cancer cells that bind to the EpRE as well as interact with the ER. One copy of EpRE (representing the −476 and −446 region of the QR gene) was cloned upstream of the LacZ and HIS3 reporter gene promoter followed by genomic integration of reporter constructs into yeast cells. cDNA libraries from MCF7 breast cancer cells were screened for expression of putative EpRE-interacting clones by their ability to activate LacZ and HIS3 reporter gene constructs. To verify the interaction of these clones with the EpRE we mutated the 5-base pair core sequence that is well conserved among the EpREs identified from different genes. Because protein factors that modulate the transcriptional activation of the QR gene by antiestrogens most likely interact with the ER, putative EpRE-interacting clones were also screened for their ability to interact with the ER. Clones were introduced into yeast cells expressing the EF domain of human ERβ fused to the GAL4 DNA binding domain, and their interaction was measured by the ability of the clones to activate a reporter construct containing the GAL4 upstream activating sequence. Of eight putative EpRE interacting clones identified, one clone was selected for further analyses because of strong binding to the EpRE and interaction with ERβ in two separate yeast screenings (Fig.1). The sequence of the putative EpRE- and ER-interacting clone was determined, and GenBank™ and literature searches indicated that the clone was the human homologue of the X enopus laevis gene, which prevents mitoticcatastrophe, XPMC2 (18Kwiatkowska J. Slomski R. Jozwiak S. Short M.P. Kwiatkowski D.J. Genomics. 1997; 44: 350-354Crossref PubMed Scopus (6) Google Scholar). The partial hPMC2 clone obtained from yeast two hybrid screening contains amino acids 139–422 of human PMC2. cDNA encoding amino acids 1–138 was obtained using reverse transcriptase-PCR and fused with the rest of the coding region. The full-length hPMC2 gene encodes a 47-kDa protein that prevents premature entry into mitosis (termed mitotic catastrophe) in fission yeast (19Su J.Y. Maller J.L. Mol. Gen. Genet. 1995; 246: 387-396Crossref PubMed Scopus (20) Google Scholar). hPMC2 has 422 amino acids and is basic with many lysine residues. An exonuclease domain has been identified at the carboxyl-terminal region (amino acids 233–406 (20Moser M.J. Holley W.R. Chatterjee A. Mian I.S. Nucleic Acids Res. 1997; 25: 5110-5118Crossref PubMed Scopus (201) Google Scholar)). hPMC2 also has a P-loop motif (Gly-X-Gly-X-X-Gly) with a well conserved lysine at amino acid residues 252–257, indicative of an ATP binding domain (21Saraste M. Sibbald P.R. Wittinghofer A. Trends Biochem. Sci. 1990; 15: 430-434Abstract Full Text PDF PubMed Scopus (1727) Google Scholar). Several potential phosphorylation sites for protein kinase C are localized from amino acids 9 to 98, as well as a tyrosine phosphorylation site at amino acids 342–350. The carboxyl-terminal region of hPMC2 also shows homology to two other known proteins: HEM45, an estrogen regulated transcript in human tumor lines and rat uterus (22Pentecost B.T. J. Steroid Biochem. Mol. Biol. 1998; 64: 25-33Crossref PubMed Scopus (31) Google Scholar), and ISG20, an interferon-induced promyelocytic leukemia protein nuclear body-associated protein (23Gongora C. David G. Pintard L. Tissot C. Hua T.D. Dejean A. Mechti N. J. Biol. Chem. 1997; 272: 19457-19463Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The interaction of hPMC2 with the ERα and ERβ was examined in vitro in GST pull-down assays. In vitro translated and radiolabeled ERβ was retained in a column wherein hPMC2 was expressed as a fusion protein with GST bound to a glutathione-Sepharose, indicating a direct interaction between hPMC2 and ERβ (Fig. 2). The interaction of hPMC2 with ERβ appears to be stronger when compared with its interaction to ERα. With regard to the effects of ligand on the interaction of ERβ with hPMC2, the strength of interaction of hPMC2 with ERβ can be described as follows: tamoxifen-liganded ERβ = unliganded ERβ > estrogen-liganded ERβ. To verify binding of hPMC2 to the EpRE, the interaction of purified FLAG-hPMC2 protein (Fig. 3 A) with radiolabeled EpRE was examined in gel shift assays. A shifted DNA-protein complex is evident in samples containing purified hPMC2 but not with control extracts (Fig. 3 B). Binding of hPMC2 to the EpRE occurred in a dose-dependent manner. The DNA-protein complex was competed by excess amounts of unlabeled EpRE but not mutated EpRE or wild type ERE (Fig. 3 C). A supershifted complex was observed in the presence of FLAG antibody. These studies indicate that hPMC2 interacted with the EpRE in a specific manner. Because our GST pull-down assays indicate the hPMC2 interacts with ERβ and more strongly so than with ERα, we examined the effect of purified ERβ (Fig. 3 A) on the ability of hPMC2 to bind to the EpRE. When purified hPMC2 was coincubated with purified ERβ, we observed a more intense DNA-protein complex band as well as supershifted bands when compared with hPMC2 alone (Fig. 3 D). Increased binding was observed in the absence of ligand and in the presence of the antiestrogen TOT. These results suggest an enhancement of the ability of hPMC2 to bind to the EpRE in the presence of ERβ. The DNA-protein complex observed in the presence of hPMC2 and ERβ was slightly supershifted and immunodepleted in the presence of ERβ antibody (Fig. 3 D), suggesting that an interaction between ERβ and hPMC2 is involved in the enhancement of hPMC2 binding to the EpRE. The ability of hPMC2 to bind to the EpRE as well as interact with the ER is supported by experiments examining intracellular localization of hPMC2. Using fluorescence microscopy, we observed that transiently transfected GFP-tagged hPMC2 can be localized primarily in the nucleus (Fig. 4). GFP vector alone showed cytoplasmic and nuclear localization, and GFP-tagged prenylcysteine carboxyl methyltransferase that has been reported to exhibit primarily non-nuclear localization (24Dai Q. Choy E. Chiu V. Romano J. Slivka S.R. Steitz S.A. Michaelis S. Philips M.R. J. Biol. Chem. 1998; 273: 15030-15034Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar) did so under our experimental conditions. To determine if binding of hPMC2 to the EpRE results in the regulation of QR gene transcriptional activity in mammalian cells, hPMC2 cDNA was cloned into the pCMV-Tag2B mammalian expression vector and cotransfected with a CAT reporter construct containing the EpRE (wild type or mutated) cloned upstream of the heterologous TK promoter. These experiments were performed in ER-negative MDA-MB-231 breast cancer cells and HepG2 liver carcinoma because of the low basal or non-induced levels of QR in these cells. Increasing amounts of hPMC2 expression vector induced a slight, albeit statistically significant increase (1.7 ± 0.2, p < 0.01) in EpRE-TK-CAT activity in MDA-MB-231 cells (Fig. 5 A). This represents maximal activation because further increases in the amount of hPMC2 expression vector did not increase reporter activity. Because increased hPMC2 binding to the EpRE was observed in the presence of ERβ, we determined if ERβ could enhance the activation of QR transcriptional activity by hPMC2. For these experiments we transfected levels of expression vector for ERβ that did not induce activation of EpRE-tk-CAT activity. Under these conditions we observe a more significant activation (3.2 ± 0.3) of EpRE enhancer activity. This level of induction is comparable with that observed withte
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