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• W2141283740 abstract "Expression of the DNA topoisomerase IIα (topoIIα) gene is highly sensitive to various environmental stimuli including heat shock. The amount of topoIIα mRNA was increased 1.5–3-fold 6–24 h after exposure of T24 human urinary bladder cancer cells to heat shock stress at 43 °C for 1 h. The effect of heat shock on the transcriptional activity of the human topoIIα gene promoter was investigated by transient transfection of T24 cells with luciferase reporter plasmids containing various lengths of the promoter sequence. The transcriptional activity of the full-length promoter (nucleotides (nt) −295 to +85) and of three deletion constructs (nt −197 to +85, −154 to +85, and −74 to +85) was increased ∼3-fold 24 h after heat shock stress. In contrast, the transcriptional activity of the minimal promoter (nt −20 to +85), which lacks the first inverted CCAAT element (ICE1), the GC box, and the heat shock element located between nt −74 and −21, was not increased by heat shock. Furthermore, the transcriptional activity of promoter constructs containing mutations in the GC box or heat shock element, but not that of a construct containing mutations in ICE1, was significantly increased by heat shock. Electrophoretic mobility shift assays revealed reduced binding of a nuclear factor to an oligonucleotide containing ICE1 when nuclear extracts were derived from cells cultured for 3–24 h after heat shock. No such change in factor binding was apparent with an oligonucleotide containing the heat shock element of the topoIIα gene promoter. Finally, in vivo footprint analysis of the topoIIα gene promoter revealed that two G residues of ICE1 that were protected in control cells became sensitive to dimethyl sulfate modification after heat shock. These results suggest that transcriptional activation of the topoIIα gene by heat shock requires the release of a negative regulatory factor from ICE1. Expression of the DNA topoisomerase IIα (topoIIα) gene is highly sensitive to various environmental stimuli including heat shock. The amount of topoIIα mRNA was increased 1.5–3-fold 6–24 h after exposure of T24 human urinary bladder cancer cells to heat shock stress at 43 °C for 1 h. The effect of heat shock on the transcriptional activity of the human topoIIα gene promoter was investigated by transient transfection of T24 cells with luciferase reporter plasmids containing various lengths of the promoter sequence. The transcriptional activity of the full-length promoter (nucleotides (nt) −295 to +85) and of three deletion constructs (nt −197 to +85, −154 to +85, and −74 to +85) was increased ∼3-fold 24 h after heat shock stress. In contrast, the transcriptional activity of the minimal promoter (nt −20 to +85), which lacks the first inverted CCAAT element (ICE1), the GC box, and the heat shock element located between nt −74 and −21, was not increased by heat shock. Furthermore, the transcriptional activity of promoter constructs containing mutations in the GC box or heat shock element, but not that of a construct containing mutations in ICE1, was significantly increased by heat shock. Electrophoretic mobility shift assays revealed reduced binding of a nuclear factor to an oligonucleotide containing ICE1 when nuclear extracts were derived from cells cultured for 3–24 h after heat shock. No such change in factor binding was apparent with an oligonucleotide containing the heat shock element of the topoIIα gene promoter. Finally, in vivo footprint analysis of the topoIIα gene promoter revealed that two G residues of ICE1 that were protected in control cells became sensitive to dimethyl sulfate modification after heat shock. These results suggest that transcriptional activation of the topoIIα gene by heat shock requires the release of a negative regulatory factor from ICE1. DNA topoisomerases are essential enzymes that participate in the segregation of newly replicated chromosome pairs, in chromosome condensation, and in modification of the superhelical content of DNA (1Berger J.M. Gamblin S.J. Harrison S.C. Wang J.C. Nature. 1996; 379: 225-232Crossref PubMed Scopus (747) Google Scholar, 2Wang J.C. Annu. Rev. Biochem. 1985; 54: 665-697Crossref PubMed Scopus (1644) Google Scholar, 3Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2086) Google Scholar). Human topoisomerase II (topoII) 1The abbreviations used are: topoII, topoisomerase II; ICE, inverted CCAAT element; HSE, heat shock element; PCR, polymerase chain reaction; nt, nucleotide(s); EMSA, electrophoretic mobility shift assay; HSF, heat shock factor. functions as a homodimer by cleaving and opening one DNA duplex, passing a second duplex through the opening, and then resealing the break (4Brown P.O. Peebles C.L. Cozzarelli N.R. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 6110-6119Crossref PubMed Scopus (117) Google Scholar, 5Gellert M. Annu. Rev. Biochem. 1981; 50: 879-910Crossref PubMed Scopus (858) Google Scholar, 6Liu L.F. Liu C.C. Alberts B.M. Cell. 1980; 19: 697-707Abstract Full Text PDF PubMed Scopus (396) Google Scholar). Two topoII isoforms have been identified in mammals: 170-kDa topoIIα and 180-kDa topoIIβ (7Jenkins J.R. Ayton P. Jones T. Davies S.L. Simmons D.L. Harris A.L. Sheer D. Hickson I.D. Nucleic Acids Res. 1992; 20: 5587-5592Crossref PubMed Scopus (234) Google Scholar). Although both enzymes are closely related in structure, they differ in important biochemical and pharmacological properties, including sensitivity to topoII-targeting drugs, cellular localization, and regulation by the cell cycle (8Drake F.H. Hofmann G.A. Bartus H.F. Mattern M.R. Crooke S.T. Mirabelli C.K. Biochemistry. 1989; 28: 8154-8160Crossref PubMed Scopus (423) Google Scholar). Whereas the amount of topoIIβ remains relatively constant throughout the cell cycle, topoIIα expression is coupled to the cell cycle (9Woessner R.D. Mattern M.R. Mirabelli C.K. Johnson R.K. Drake F.H. Cell Growth Differ. 1991; 2: 209-214PubMed Google Scholar, 10Goswami P.C. Roti Roti J.L. Hunt C.R. Mol. Cell. Biol. 1996; 16: 1500-1508Crossref PubMed Google Scholar). topoIIα is of particular importance because of its association with DNA replication, mitosis, and cell proliferation. Expression of topoIIα is highly susceptible to environmental stimuli, and such regulation is thought to be mediated at both the transcriptional and post-transcriptional levels. The promoter region of the topoIIα gene contains various regulatory elements, including five inverted CCAAT elements (ICEs), one GC box, and one heat shock element (HSE) (11Hochhauser D. Stanway C.A. Harris A.L. Hickson I.D. J. Biol. Chem. 1992; 267: 18961-18965Abstract Full Text PDF PubMed Google Scholar). Exposure of human colon cancer cells to glucosamine induces down-regulation of topoIIα, resulting in the development of resistance to the topoIIα-targeting epipodophyllotoxin, etoposide (12Yun J. Tomida A. Nagata K. Tsuruo T. Oncol. Res. 1995; 7: 583-590PubMed Google Scholar). Development of resistance to such topoIIα-targeting agents is often associated with down-regulation of topoIIα in various mammalian cell lines (13Takano H. Kohno K. Matsuo K. Matsuda T. Kuwano M. Anti-Cancer Drugs. 1992; 3: 323-330Crossref PubMed Scopus (72) Google Scholar, 14Beck J. Niethammer D. Gekeler V. Cancer Lett. 1994; 86: 135-142Crossref PubMed Scopus (48) Google Scholar). In one etoposide-resistant cell line derived from human head and neck cancer KB cells (15Takano H. Kohno K. Ono M. Uchida Y. Kuwano M. Cancer Res. 1991; 51: 3951-3957PubMed Google Scholar, 16Matsuo K. Kohno K. Takano H. Sato S. Kiue A. Kuwano M. Cancer Res. 1990; 50: 5819-5824PubMed Google Scholar), the transcription factor Sp3 was implicated in the down-regulation of topoIIα (17Kubo T. Kohno K. Ohga T. Taniguchi K. Kawanami K. Wada M. Kuwano M. Cancer Res. 1995; 55: 3860-3864PubMed Google Scholar). Introduction of the wild-type p53 tumor suppressor gene into murine cells results in reduced expression of the topoIIα gene, and this effect appears to be mediated by one of the ICEs in the topoIIα gene promoter (18Wang Q. Zambetti G.P. Suttle D.P. Mol. Cell. Biol. 1997; 17: 389-397Crossref PubMed Google Scholar). Apoptosis induced by adenovirus E1A protein in human KB cells is associated with a marked decrease in the amount of topoIIα that is due to accelerated degradation of topoIIα by the ubiquitin proteolysis pathway (19Nakajima T. Ohi N. Arai T. Nozaki N. Kikuchi A. Oda K. Oncogene. 1995; 10: 651-662PubMed Google Scholar, 20Nakajima T. Morita K. Ohi N. Arai T. Nozaki N. Kikuchi A. Osaka F. Yamao F. Oda K. J. Biol. Chem. 1996; 271: 24842-24849Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The amount of topoIIα mRNA in late S phase is ∼15 times that during the G1 phase of the cell cycle in human HeLa cells, apparently because of increased mRNA stability in S phase (10Goswami P.C. Roti Roti J.L. Hunt C.R. Mol. Cell. Biol. 1996; 16: 1500-1508Crossref PubMed Google Scholar). These observations indicate that topoIIα expression is regulated by multiple mechanisms that operate at the levels of transcription, mRNA stability, and protein degradation. Heat shock stress also affects the abundance of topoIIα mRNA in mammalian cells. Exposure of human head and neck or colon cancer cells to high nonpermissive temperatures results in an increase in expression of the topoIIα gene, apparent 6–12 h later, and consequent sensitization to the cytotoxic effect of etoposide (21Brandt T.L. Fraser D.J. Leal S. Halandras P.M. Kroll A.R. Kroll D.J. J. Biol. Chem. 1997; 272: 6278-6284Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 22Matsuo K. Kohno K. Sato S. Uchiumi T. Tanimura H. Yamada Y. Kuwano M. Cancer Res. 1993; 53: 1085-1090PubMed Google Scholar). The same heat shock stress markedly increases the abundance of the heat shock protein HSP70 and induces a transient decrease in the amount of topoIIα mRNA and protein immediately after exposure to hyperthermia (10Goswami P.C. Roti Roti J.L. Hunt C.R. Mol. Cell. Biol. 1996; 16: 1500-1508Crossref PubMed Google Scholar, 22Matsuo K. Kohno K. Sato S. Uchiumi T. Tanimura H. Yamada Y. Kuwano M. Cancer Res. 1993; 53: 1085-1090PubMed Google Scholar, 23Hirohashi Y. Hidaka K. Sato S. Kuwano M. Kohno K. Hisatsugu T. Jpn. J. Cancer Res. 1995; 86: 1097-1105Crossref PubMed Scopus (7) Google Scholar). Whereas this early effect of heat shock stress on topoIIα expression appears to be mediated by increased degradation of topoIIα mRNA (10Goswami P.C. Roti Roti J.L. Hunt C.R. Mol. Cell. Biol. 1996; 16: 1500-1508Crossref PubMed Google Scholar), the later up-regulation of topoIIα gene expression appears to be due to transcriptional activation (22Matsuo K. Kohno K. Sato S. Uchiumi T. Tanimura H. Yamada Y. Kuwano M. Cancer Res. 1993; 53: 1085-1090PubMed Google Scholar). We have now investigated which elements in the 5′-flanking region of the human topoIIα gene are responsible for the heat shock-induced activation of transcription. Restriction enzymes and other nucleic acid-modifying enzymes and reagents were obtained from Promega (Madison, WI), Life Technologies, Inc., or Takara Shuzo (Kyoto, Japan), unless indicated otherwise. Both [α-32P]dCTP and [γ-32P]ATP were from NEN Life Science Products. Human topoI cDNA was kindly provided by T. Andoh (Sohka University, Tokyo, Japan), and human topoIIα cDNA (pBS-hTOP2) was provided by J. C. Wang (Harvard University, Boston, MA). Human HSP70 cDNA was kindly given by R. T. N. Tjian (University of California, Berkeley, CA). All cDNA fragments were separated from vector DNA by agarose gel electrophoresis and labeled by random primer DNA synthesis. The T24 cell line, established from human transitional cell carcinoma of the urinary bladder (24Bubenik J. Baresova M. Viklicky V. Jakoubkova J. Sainerova H. Donner J. Int. J. Cancer. 1973; 11: 765-773Crossref PubMed Scopus (472) Google Scholar), was cultured at 37 °C under a humidified atmosphere of 5% CO2 in Eagle's minimal essential medium (Nissui Seiyaku, Tokyo) supplemented with 10% newborn calf serum (Sera-Lab, Sussex, United Kingdom), 1 mg/ml Bacto-peptone (Difco), 0.292 mg/mll-glutamine, 100 units/ml penicillin, and 100 μg/ml kanamycin. For heat shock, culture plates were sealed with paraffin film and immersed in a water bath at 43 °C for 1 h. Northern blot analysis was performed as described previously (17Kubo T. Kohno K. Ohga T. Taniguchi K. Kawanami K. Wada M. Kuwano M. Cancer Res. 1995; 55: 3860-3864PubMed Google Scholar). Briefly, total RNA was extracted from T24 cells with the use of guanidine isothiocyanate (25Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar), subjected (15 μg/lane) to electrophoresis on a 1% agarose gel containing formaldehyde, and transferred to a Hybond N+ membrane (Amersham International, Buckinghamshire, United Kingdom). The membranes were exposed to 32P-labeled cDNA probes for 18 h and washed twice at 42 °C in 2× SSC containing 0.1% SDS and twice at 42 °C in 0.2× SSC containing 0.1% SDS. Radioactivity was detected with a Fujix BAS 2000 image analyzer (Fuji Film, Tokyo). We used the polymerase chain reaction (PCR) to clone the human topoIIα gene promoter (nt −295 to +85, relative to the major transcription start site) as described previously (17Kubo T. Kohno K. Ohga T. Taniguchi K. Kawanami K. Wada M. Kuwano M. Cancer Res. 1995; 55: 3860-3864PubMed Google Scholar). The 3′-end of all inserts was nt +85, 10 base pairs upstream of the translation initiation site. For the construction of other deletion constructs, HindIII fragments (nt −295 to +85) of the pTIIα−295 plasmid were digested with BfaI (pTIIα−197), ScrFI (pTIIα−154),HphI (pTIIα−74), and SacI (pTIIα−20). The digestion products were blunt-ended with the Klenow fragment of DNA polymerase I, ligated to HindIII linkers, and cloned into the HindIII site of the pGL2-Basic vector (Promega). Site-directed mutagenesis of ICE1, the GC box, and the HSE in pTIIα−295 was performed by a PCR-based method. The promoter sequences were amplified first with Pfu polymerase (Stratagene, La Jolla, CA), the 3′-primer +85 (5′-CGGTCGTGAAGGGGCTCAAG-3′), and 5′-primers that introduce specific mutations into the target elements: m5 (5′-CAGGGAAAAACTGGTCTGCTTCGGGCGGGCTAAAGGAAGGTTCAAGTGGAGCT-3′) for mutation of ICE1, m6 (5′-CAGGGATTGGCTGGTCTGCTTCAAAAAAGCTAAAGGAAGGTTCAAGTGGAGCT-3′) for mutation of the GC box, and m7 (5′-CAGGGATTGGCTGGTCTGCTTCGGGCGGGCTAAAGAAAGGAAAAAATGGAGCT-3′) for mutation of the HSE (mutated nucleotides are underlined). A second PCR was then performed with Taq polymerase, the first PCR products, and the 5′-primer −295 (corresponding to the normal promoter sequence with a 5′-end at nt −295). The second PCR products were digested with HindIII and ligated into pGL2-Basic. The mutations introduced into these clones were confirmed by DNA sequencing. T24 cells (1 × 105) were transferred to 60-mm dishes, incubated at 37 °C for 48 h, and transfected with luciferase plasmid DNA (2.5 μg) by calcium phosphate precipitation as described previously (26Okimoto T. Kohno K. Kuwano M. Gopas J. Kung H.F. Ono M. Oncogene. 1996; 12: 1625-1633PubMed Google Scholar). Four hours after transfection, the cells were washed, incubated at 37 °C for 24 h in fresh medium, and exposed to 43 °C for 1 h. The treated cells were then harvested immediately (0 h) or after further incubation at 37 °C for 1, 6, 12, or 24 h for determination of luciferase activity. Cells were lysed in 200 μl of 25 mm Tris phosphate buffer (pH 7.5) containing 1% Triton X-100 and subjected to centrifugation at 14,000 × gfor 15 s. The resulting supernatants were assayed for luciferase activity with the use of a Picagene kit (Toyoinki, Tokyo); light intensity was measured for 15 s with a luminometer (Model TD-20/20, Promega). Cells were cotransfected with pSV2-β-GAL as a control for transfection efficiency, and β-galactosidase activity was measured with an Aurora GAL-XE kit (ICN, Costa Mesa, CA). Heat-treated or control T24 cells were exposed to dimethyl sulfate, and genomic DNA was then extracted and cleaved as described (27Konishi T. Nomoto M. Shimizu K. Abe T. Itoh H. J. Biochem. (Tokyo). 1995; 118: 1021-1029Crossref PubMed Scopus (24) Google Scholar, 28Abravaya K. Phillips B. Morimoto R.I. Mol. Cell. Biol. 1991; 11: 586-592Crossref PubMed Scopus (125) Google Scholar). Ligation-mediated PCR was performed as described (27Konishi T. Nomoto M. Shimizu K. Abe T. Itoh H. J. Biochem. (Tokyo). 1995; 118: 1021-1029Crossref PubMed Scopus (24) Google Scholar). Primer 1 (5′-CAGGCAGGACCCCACG-3′, nt +46 to +31) was used for first-strand synthesis; primer 2 (5′-CCCGACCAAGCCGCTTCTCCAC-3′, nt +22 to +1) was used for PCR amplification; and primer 3 (5′-CCGACCAAGCCGCTTCTCCACAGACGCG-3′, nt +21 to −7), which was labeled at the 5′-end with [γ-32P]ATP and T4 polynucleotide kinase, was used for final detection of the DNA ladder. Samples were analyzed on a 6% polyacrylamide sequencing gel. T24 cells (5 × 105) were transfected with a luciferase reporter vector containing the topoIIα gene promoter (pTIIα−295; 10 μg) and pRSV-neo (0.5 μg) with the use of Trans-it reagent (PanVera, Madison, WI). After 8 h, the medium was replaced, and the cells were incubated for 24 h. The cells were then incubated in selection medium containing G418 (0.8 mg/ml; Life Technologies, Inc.), and growing colonies (20–30/106 cells) were cloned, expanded, and tested for luciferase activity. Unless indicated otherwise, PCR was performed in a final volume of 100 μl containing 1 ng of template DNA, a 100 pm concentration of each oligonucleotide primer, a 200 μm concentration of each deoxynucleotide triphosphate, 2.5 units of Taq DNA polymerase, 50 mm KCl, 10 mm Tris-HCl (pH 8.3), 1.5 mm MgCl2, and 0.01% (w/v) gelatin. Amplification was carried out in a DNA thermal cycler (Perkin-Elmer) for 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 1 min, and polymerization at 72 °C for 2 min. Nuclear extracts were prepared as described previously (17Kubo T. Kohno K. Ohga T. Taniguchi K. Kawanami K. Wada M. Kuwano M. Cancer Res. 1995; 55: 3860-3864PubMed Google Scholar). Briefly, T24 cells (4 × 107), subjected or not to heat shock at 43 °C for 1 h, were collected by exposure to trypsin; resuspended in 200 μl of an ice-cold solution containing 10 mm Hepes-NaOH (pH 7.9), 10 mm KCl, 0.75 mm spermidine, 0.15 mmspermine, 0.2 mm EDTA, 0.2 mm EGTA, 0.5 mm dithiothreitol, and 0.5 mmphenylmethylsulfonyl fluoride; and incubated on ice for 15 min. The cells were then lysed by passing 10 times through a 25-gauge needle attached to a 1-ml syringe, and the lysate was centrifuged for 40 s in a microcentrifuge. The resulting nuclear pellet was resuspended in 100 μl of an ice-cold solution containing 20 mmHepes-NaOH (pH 7.9), 0.4 m NaCl, 0.75 mmspermidine, 0.15 mm spermine, 0.2 mm EDTA, 0.2 mm EGTA, 0.5 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, and 25% (v/v) glycerol; incubated for 30 min on ice with frequent gentle mixing; and then centrifuged for 20 min at 4 °C in a microcentrifuge to remove insoluble material. The resulting supernatant (nuclear extract) was stored at −70 °C, and its protein concentration was determined with a protein assay kit (Bio-Rad). EMSAs were performed as described previously (29Miyazaki M. Kohno K. Uchiumi T. Tanimura H. Matsuo K. Nasu M. Kuwano M. Biochem. Biophys. Res. Commun. 1992; 187: 677-684Crossref PubMed Scopus (89) Google Scholar). Briefly, 6 μg of nuclear extract protein were incubated for 30 min at room temperature in a final volume of 20 μl containing 10 mm Tris-HCl (pH 7.5), 50 mm NaCl, 1 mm MgCl2, 1 mm EDTA, 8% glycerol, 1 mm dithiothreitol, 0.1 μg of poly(dI-dC), and 1 × 104 cpm of32P-labeled oligonucleotide probe (1 ng) in the absence or presence of various competitors. The reaction mixtures were then applied to a nondenaturing 5% polyacrylamide gel and separated by electrophoresis at 100 V for 3 h in a buffer containing 50 mm Tris, 380 mm glycine, and 2 mmEDTA. The gel was exposed to x-ray film with intensifying screens. The following oligonucleotides were used for EMSAs: topo-ICE1 (5′-GAGTCAGGGATTGG CTGGTCTGCTTCGGGC-3′, nt −77 to −48 of the topoIIα gene), topo-HSE (5′-GGGCTAAAGG AAGGTTCAAGTGGAGCTCTC-3′, nt −47 to −18 of the topoIIα gene), and HSP70-HSE (5′-GA AACCCCTGGAATATTCCCGACC-3′, nt −114 to −91 of the human HSP70 gene). For supershift assays, 2 μg of antibodies to heat shock factor HSF1 or HSF2 (30Nakai A. Kawazoe Y. Tanabe M. Nagata K. Morimoto R.I. Mol. Cell. Biol. 1995; 15: 5268-5278Crossref PubMed Scopus (90) Google Scholar) were incubated with nuclear extract for 30 min at room temperature before addition of32P-labeled oligonucleotide probe. Consistent with our previous observations with human head and neck or colorectal cancer cells (22Matsuo K. Kohno K. Sato S. Uchiumi T. Tanimura H. Yamada Y. Kuwano M. Cancer Res. 1993; 53: 1085-1090PubMed Google Scholar, 23Hirohashi Y. Hidaka K. Sato S. Kuwano M. Kohno K. Hisatsugu T. Jpn. J. Cancer Res. 1995; 86: 1097-1105Crossref PubMed Scopus (7) Google Scholar), Northern blot analysis revealed that exposure of T24 cells to 43 °C for 1 h resulted in an initial small decrease in the amount of topoIIα mRNA, which was followed by an increase in transcript abundance that was maximal (∼3-fold) at 24 h (Fig. 1). The amount of HSP70mRNA was increased immediately after heat treatment, reaching a maximum (∼18-fold induction) at 1 h. In contrast, the amount of topoI mRNA was not affected by heat stress. The HSP70and topoIIα genes thus showed characteristics of immediate-early and late genes, respectively, in response to heat shock. We measured the basal transcriptional activity of the topoIIα gene promoter in T24 cells transiently transfected with various luciferase reporter plasmids (Fig. 2). Maximal luciferase activity was obtained with the reporter construct with the pTIIα−295 insert, which contains four ICEs, the GC box, and the HSE between nt −295 and +85 of the topoIIα gene. Stepwise deletion of ICE3, ICE2, and the combination of ICE1, GC box, and HSE from the 5′-end of the promoter resulted in marked -fold decreases in luciferase activity, in general agreement with previous results (11Hochhauser D. Stanway C.A. Harris A.L. Hickson I.D. J. Biol. Chem. 1992; 267: 18961-18965Abstract Full Text PDF PubMed Google Scholar). Exposure at 43 °C for 1 h of T24 cells transiently transfected with the reporter construct containing pTIIα−295 resulted in an initial ∼80% decrease in luciferase activity, followed by an increase that was maximal (3-fold) 24 h after heat treatment (Fig. 3). This experiment was repeated with two T24 cell lines stably transfected with the pTIIα−295 luciferase construct. Again, luciferase activity was decreased immediately after heat treatment, but then showed a time-dependent increase that was maximal (3–4-fold) after 24 h (data not shown). To identify the promoter sequences responsible for conferring sensitivity to heat shock, we measured luciferase activity 24 h after exposure to 43 °C for 1 h of T24 cells transiently transfected with various topoIIα gene promoter constructs (Fig. 4). Heat shock increased luciferase activity ∼3-fold in cells transfected with pTIIα−295, pTIIα−197, pTIIα−154, or pTIIα−74, but did not increase luciferase activity in cells transfected with pTIIα−20. The promoter sequence between nt −74 and −20, which contains ICE1, the GC box, and the HSE, thus appears to mediate transcriptional activation by heat shock. The roles of ICE1, the GC box, and the HSE in heat induction of topoIIα gene promoter activity were investigated in T24 cells transiently transfected with luciferase reporter plasmids containing promoter sequences with specific mutations in these elements: GGATTGGCT in ICE1 was converted to GGAAAAACT (pTIIα−295m5), GGGCGGG in the GC box to AAAAAAG (pTIIα−295m6), and GGAAGGTTCAAGTG in the HSE to GAAAGGAAAAAATG (pTIIα−295m7) (Fig. 5 A). The pTIIα−295m5 construct showed increased basal transcriptional activity, but luciferase activity was not increased further by heat shock (Fig. 5 B). In contrast, heat shock increased the transcriptional activities of pTIIα−295m6 and pTIIα−295m7 ∼3-fold; the transcriptional activities of these two plasmids were ∼30 and 10%, respectively, of that of the wild-type plasmid. Thus, a factor that binds to ICE1 might negatively regulate basal promoter activity, and ICE1 appears to play a key role in heat-induced activation of the topoIIα gene promoter. Whereas the GC box and HSE appear to contribute to basal promoter activity, they do not appear to be directly responsible for heat-induced promoter activation. We next investigated the effects of heat shock on the ICE (Y-box) binding proteins and HSFs with the use of EMSAs. A marked decrease in Y-box binding activity was apparent 3, 6, 12, and 24 h after heat shock (Fig. 6 A). Formation of the complex was inhibited in the presence of either excess unlabeled oligonucleotide. EMSAs performed with a typical HSE derived from the human HSP70.1 gene revealed the absence of a retarded signal in untreated cells (Fig. 6 B). A retarded complex was detected with nuclear extracts of cells prepared immediately (0 h) after heat treatment; formation of this complex was inhibited in the presence of excess unlabeled oligonucleotide, and the complex was “supershifted” in the presence of antibodies to HSF1, but not in the presence of antibodies to HSF2. With the HSE of the topoIIα gene as probe, a retarded complex was observed with nuclear extracts prepared from untreated cells and from cells after heat shock (Fig. 6 C). However, the amount of this retarded complex was not affected by heat stress. Formation of this complex was inhibited by excess unlabeled oligonucleotide, but was not affected by antibodies to HSF1 or HSF2. We examined the effects of heat shock on the dimethyl sulfate methylation patterns in the promoter region of the topoIIα gene by in vivo genomic footprint analysis. Both G−64 and G−65 in ICE1 were protected in untreated cells, but protection was markedly reduced 3, 6, and 24 h after heat shock (Fig. 7). Methylation patterns of the GC box, HSE, and other elements in the topoIIα promoter region (nt −295 to +85) were not substantially affected by heat shock stress (data not shown). We have previously shown that expression of the topoIIα gene is increased 6–24 h after exposure of human head and neck or colorectal cancer cells to heat shock stress (22Matsuo K. Kohno K. Sato S. Uchiumi T. Tanimura H. Yamada Y. Kuwano M. Cancer Res. 1993; 53: 1085-1090PubMed Google Scholar, 23Hirohashi Y. Hidaka K. Sato S. Kuwano M. Kohno K. Hisatsugu T. Jpn. J. Cancer Res. 1995; 86: 1097-1105Crossref PubMed Scopus (7) Google Scholar). In the present study, we have shown that heat stress also induced activation of topoIIα gene expression in human urinary bladder cancer cells. This heat-induced up-regulation of topoIIα gene expression appeared to be mediated through an ICE or Y-box located between nt −74 and −21 on the basis of the following results. (i) The luciferase activity of T24 cells transfected with reporter constructs containing pTIIα−295, pTIIα−197, pTIIα−154, or pTIIα−74 was increased ∼3-fold by heat shock stress, whereas that of cells transfected with a construct containing pTIIα−20 was not increased by heat treatment. (ii) Introduction of mutations into ICE1 of the topoIIα gene promoter virtually eliminated the heat shock-induced increase in transcriptional activity, whereas mutation of the GC box or HSE had no such effect. (iii) EMSA analysis with nuclear extracts revealed a marked decrease in ICE1-binding activity 3–24 h after heat shock, consistent with the time course of the heat shock-induced increase in promoter activity, whereas HSE-binding activity was not affected by heat stress. (iv)In vivo genomic footprint analysis revealed a specific change in the methylation pattern of ICE1 induced by heat shock stress. Members of the ICE-binding (YB-1) family of proteins are expressed in a wide range of cell types and function as important regulators of growth-associated and other genes (31Ladomery M. Sommerville J. Bioessays. 1995; 17: 9-11Crossref PubMed Scopus (130) Google Scholar, 32Ohga T. Koike K. Ono M. Makino Y. Itagaki Y. Tanimoto M. Kuwano M. Kohno K. Cancer Res. 1996; 56: 4224-4228PubMed Google Scholar, 33Wolffe A.P. Tafuri S. Ranjan M. Familari M. New Biol. 1992; 4: 290-298PubMed Google Scholar, 34Wolffe A.P. Bioessays. 1994; 16: 245-251Crossref PubMed Scopus (329) Google Scholar). The expression of genes encoding the epidermal growth factor receptor (35Sakura H. Maekawa T. Imamoto F. Yasuda K. Ishii S. Gene (Amst.). 1988; 73: 499-507Crossref PubMed Scopus (146) Google Scholar), proliferating cell nuclear antigen (36Travali S. Ku D.H. Rizzo M.G. Ottavio L. Baserga R. Calabretta B. J. Biol. Chem. 1989; 264: 7466-7472Abstract Full Text PDF PubMed Google Scholar), DNA polymerase α (37Pearson B.E. Nasheuer H.P. Wang T.S. Mol. Cell. Biol. 1991; 11: 2081-2095Crossref PubMed Google Scholar), and thymidine kinase (38Lipson K.E. Chen S.T. Koniecki J. Ku D.H. Baserga R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6848-6852Crossref PubMed Scopus (42) Google Scholar) is regulated in a positive manner by ICEs. In contrast, such elements mediate down-regulation of the expression of genes encoding serum albumin, estrogen-dependent very low density lipoprotein apolipoprotein II, aldolase B, and class II major histocompatibility complex (39Grant C.E. Deeley R.G. Mol. Cell. Biol. 1993; 13: 4186-4196Crossref PubMed Scopus (83) Google Scholar, 40Ito K. Tsutsumi K. Kuzumaki T. Gomez P.F. Otsu K. Ishikawa K. Nucleic Acids Res. 1994; 22: 2036-2041Crossref PubMed Scopus (53) Google Scholar, 41Ting J.P. Painter A. Zeleznik-Le N.J. MacDonald G. Moore T.M. Brown A. Schwartz B.D. J. Exp. Med. 1994; 179: 1605-1611Crossref PubMed Scopus (79) Google Scholar). In the present study, deletion of nt −197 to −155, with contain ICE3, reduced basal promoter activity to about half of that apparent with the topoIIα gene promoter constructs pTIIα−295 and pTIIα−197. Further deletion of nt −154 to −75, containing ICE2, and of nt −74 to −21, containing ICE1, reduced basal promoter activity to ∼10 and 2%, respectively, of that apparent with pTIIα−295. Consecutive deletion of the five ICEs from the topoIIα gene promoter was also previously shown to reduce basal promoter activity in a stepwise manner (11Hochhauser D. Stanway C.A. Harris A.L. Hickson I.D. J. Biol. Chem. 1992; 267: 18961-18965Abstract Full Text PDF PubMed Google Scholar, 18Wang Q. Zambetti G.P. Suttle D.P. Mol. Cell. Biol. 1997; 17: 389-397Crossref PubMed Google Scholar). Thus, the ICEs in the promoter of the human topoIIα gene appear to play an important role in basal transcriptional activity. Introduction of point mutations into ICE1 of the topoIIα gene promoter alleviated the inhibition of topoIIα gene expression by wild-type p53 (18Wang Q. Zambetti G.P. Suttle D.P. Mol. Cell. Biol. 1997; 17: 389-397Crossref PubMed Google Scholar). Fraser et al. (42Fraser D.J. Brandt T.L. Kroll D.J. Mol. Pharmacol. 1995; 47: 696-706PubMed Google Scholar) showed that the topoIIα gene promoter is activated at an early stage during monocytic differentiation of human leukemia cells induced by phorbol ester or sodium butyrate and that this sodium butyrate-dependent up-regulation of topoIIα gene expression is mediated by the promoter region between nt −90 and +90, which contains ICE1. In contrast, inhibition of topoIIα gene promoter activity in confluence-arrested cells appears to be mediated through interaction of the CCAAT-binding factor CBF/NF-Y with ICE2 (43Isaacs R.J. Harris A.L. Hickson I.D. J. Biol. 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Expression of YB-1 is also increased in response to genotoxic stress, suggesting that the promoter of the YB-1 gene itself is also sensitive to cytotoxic environmental stimuli (32Ohga T. Koike K. Ono M. Makino Y. Itagaki Y. Tanimoto M. Kuwano M. Kohno K. Cancer Res. 1996; 56: 4224-4228PubMed Google Scholar, 48Makino Y. Ohga T. Toh S. Koike K. Okumura K. Wada M. Kuwano M. Kohno K. Nucleic Acids Res. 1996; 24: 1873-1878Crossref PubMed Scopus (39) Google Scholar). ICEs thus appear to mediate either negative or positive regulation of specific genes in response to exogenous stimuli. Brandt et al. (21Brandt T.L. Fraser D.J. Leal S. Halandras P.M. Kroll A.R. Kroll D.J. J. Biol. Chem. 1997; 272: 6278-6284Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) recently showed that c-Myb activated the human topoIIα gene promoter via a Myb-binding site at nt −16 to −11 in human leukemia cells. In the present study, the basal promoter activity of pTIIα−20 was only 1.6% of that of pTIIα−295, and heat shock did not increase the transcriptional activity of this construct. It is thus unlikely that the Myb-binding site at −16 to −11 plays an important role in the heat activation of promoter activity of the topoIIα gene. Heat shock induces the expression of heat shock-related genes in mammalian cells, and this activation is mediated by HSFs (49Lindquist S. Annu. Rev. Biochem. 1986; 55: 1151-1191Crossref PubMed Google Scholar, 50Lis J. Wu C. Cell. 1993; 74: 1-4Abstract Full Text PDF PubMed Scopus (340) Google Scholar, 51Milarski K.L. Morimoto R.I. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9517-9521Crossref PubMed Scopus (260) Google Scholar, 52Morimoto R.I. Science. 1993; 259: 1409-1410Crossref PubMed Scopus (1240) Google Scholar, 53Schuetz T.J. Gallo G.J. Sheldon L. Tempst P. Kingston R.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6911-6915Crossref PubMed Scopus (260) Google Scholar). HSFs bind to HSEs, which consist of contiguous arrays of the pentanucleotide motif 5′-NGAAN-3′ present in alternating orientations in the promoter regions of heat shock genes. Most heat-inducible genes, including HSP genes, contain an HSE consisting of four or more pentanucleotide motifs and respond to heat treatment within 1 h concomitant with marked fluctuations in nuclear HSF content (27Konishi T. Nomoto M. Shimizu K. Abe T. Itoh H. J. Biochem. (Tokyo). 1995; 118: 1021-1029Crossref PubMed Scopus (24) Google Scholar, 30Nakai A. Kawazoe Y. Tanabe M. Nagata K. Morimoto R.I. Mol. Cell. Biol. 1995; 15: 5268-5278Crossref PubMed Scopus (90) Google Scholar,54Kroeger P.E. Sarge K.D. Morimoto R.I. Mol. Cell. Biol. 1993; 13: 3370-3383Crossref PubMed Scopus (96) Google Scholar, 55Sarge K.D. Murphy S.P. Morimoto R.I. Mol. Cell. Biol. 1993; 13: 1392-1407Crossref PubMed Scopus (754) Google Scholar). Our data confirm that HSF1, but not HSF2, binds to the HSE of the human HSP70 gene immediately after heat shock. However, the HSE of the topoIIα gene consists of only two pentanucleotide motifs, and heat shock-induced transcriptional activation of the topoIIα gene was not apparent until 6–24 h after heat treatment. Furthermore, no increase in the binding of nuclear factors to the HSE of the topoIIα gene after heat treatment was apparent by EMSA or in vivo footprint analysis. It is thus unlikely that the HSE in the topoIIα gene promoter is responsible for the heat-induced activation of this gene. We thank Takanori Nakamura (of our laboratory), Dr. Katsuhiko Hidaka (Saga Medical School), and Dr. Akira Nakai (Kyoto University) for fruitful discussion and Tomoko Matsuguma for help in preparing the manuscript." @default.
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• W2141283740 title "The Role of an Inverted CCAAT Element in Transcriptional Activation of the Human DNA Topoisomerase IIα Gene by Heat Shock" @default.
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