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• W2169071938 abstract "We have isolated two overlapping genomic clones that contain the 5′-terminal portion of the human vacuolar H+-ATPase c subunit (ATP6L) gene. The sequence preceding the transcription initiation site, which is GC-rich, contains four GC boxes and one Oct1-binding site, but there is no TATA box or CCAAT box. In vivo footprint analysis in human cancer cells shows that two GC boxes and the Oct1-binding site are occupied by Sp1 and Oct1, respectively. We show here that treatment with anticancer agents enhances ATP6Lexpression. Although cisplatin did not induce ATP6L promoter activity, it altered ATP6LmRNA stability. On the other hand, the DNA topoisomerase II inhibitor, TAS-103, strongly induced promoter activity, and this effect was completely eradicated when a mutation was introduced into the Oct1-binding site. Treatment with TAS-103 increased the levels of both Sp1/Sp3 and Oct1 in nuclear extracts. Cooperative binding of Sp1 and Oct1 to the promoter is required for promoter activation by TAS-103. Incubation of a labeled oligonucleotide probe encompassing the −73/−68 GC box and −64/−57 Oct1-binding site with a nuclear extract from drug-treated KB cells yielded higher levels of the specific DNA-protein complex than an extract of untreated cells. Thus, the two transcription factors, Sp1 and Oct1 interact, in an adaptive response to DNA damage, by up-regulating expression of the vacuolar H+-ATPase genes. Furthermore, combination of the vacuolar H+-ATPase (V-ATPase) inhibitor, bafilomycin A1, with TAS-103 enhanced apoptosis of KB cells with an associated increase in caspase-3 activity. Our data suggest that the induction of V-ATPase expression is an anti-apoptotic defense, and V-ATPase inhibitors in combination with low-dose anticancer agents may provide a new therapeutic approach. We have isolated two overlapping genomic clones that contain the 5′-terminal portion of the human vacuolar H+-ATPase c subunit (ATP6L) gene. The sequence preceding the transcription initiation site, which is GC-rich, contains four GC boxes and one Oct1-binding site, but there is no TATA box or CCAAT box. In vivo footprint analysis in human cancer cells shows that two GC boxes and the Oct1-binding site are occupied by Sp1 and Oct1, respectively. We show here that treatment with anticancer agents enhances ATP6Lexpression. Although cisplatin did not induce ATP6L promoter activity, it altered ATP6LmRNA stability. On the other hand, the DNA topoisomerase II inhibitor, TAS-103, strongly induced promoter activity, and this effect was completely eradicated when a mutation was introduced into the Oct1-binding site. Treatment with TAS-103 increased the levels of both Sp1/Sp3 and Oct1 in nuclear extracts. Cooperative binding of Sp1 and Oct1 to the promoter is required for promoter activation by TAS-103. Incubation of a labeled oligonucleotide probe encompassing the −73/−68 GC box and −64/−57 Oct1-binding site with a nuclear extract from drug-treated KB cells yielded higher levels of the specific DNA-protein complex than an extract of untreated cells. Thus, the two transcription factors, Sp1 and Oct1 interact, in an adaptive response to DNA damage, by up-regulating expression of the vacuolar H+-ATPase genes. Furthermore, combination of the vacuolar H+-ATPase (V-ATPase) inhibitor, bafilomycin A1, with TAS-103 enhanced apoptosis of KB cells with an associated increase in caspase-3 activity. Our data suggest that the induction of V-ATPase expression is an anti-apoptotic defense, and V-ATPase inhibitors in combination with low-dose anticancer agents may provide a new therapeutic approach. vacuolar H+-ATPase dithiothreitol phenylmethylsulfonyl fluoride electrophoretic mobility shift assay phosphate-buffered saline nucleotide 1,4-piperazinediethanesulfonic acid dimethyl sulfate Tumor cells possess high glycolytic activity, and rapid growth produces acidic metabolites. Moreover, tumor cells often exist in an hypoxic microenvironment lower in pH than that of surrounding normal cells. Hence, proton extrusion may be up-regulated to protect tumor cells from acidosis. Four major types of pH regulators have been identified in tumor cells as follows: sodium-proton exchangers, bicarbonate transporters, proton-lactate symporters, and proton pumps. The vacuolar H+-ATPase (V-ATPase)1 is ubiquitously expressed in eukaryotic cells (1Stevens T.H. Forgac M. Annu. Rev. Cell Dev. Biol. 1997; 13: 779-808Crossref PubMed Scopus (515) Google Scholar, 2Finbow M.E. Harrison M.A. Biochem. J. 1997; 324: 697-712Crossref PubMed Scopus (228) Google Scholar, 3Forgac M. FEBS Lett. 1998; 440: 258-263Crossref PubMed Scopus (116) Google Scholar, 4Forgac M. J. Biol. Chem. 1999; 274: 12951-12954Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 5Nishi T. Forgac M. Nat. Rev. Mol. Cell Biol. 2002; 3: 94-103Crossref PubMed Scopus (976) Google Scholar, 6Torigoe T. Izumi H. Ise T. Murakami T. Uramoto H. Ishiguchi H. Yoshida Y. Tanabe M. Nomoto M. Kohno K. Anti-Cancer Drugs. 2002; 13: 237-243Crossref PubMed Scopus (53) Google Scholar), not only in vacuolar membranes but also in plasma membrane (7Wieczorek H. Brown D. Grinstein S. Ehrenfeld J. Harvey W.R. Bioessays. 1999; 21: 637-648Crossref PubMed Scopus (223) Google Scholar, 8Merzendorfer H. Graf R. Huss M. Harvey W.R. Wieczorek H. J. Exp. Biol. 1997; 200: 225-235Crossref PubMed Google Scholar, 9Wieczorek H. Gruber G. Harvey W.R. Huss M. Merzendorfer H. Zeiske W. J. Exp. Biol. 2000; 203: 127-135Crossref PubMed Google Scholar). It is a multisubunit enzyme composed of a membrane sector and a cytosolic catalytic sector (10Bowman B.S. Dschida W.J. Harris T. Bowman E.J. J. Biol. Chem. 1989; 264: 15606-15612Abstract Full Text PDF PubMed Google Scholar); it pumps protons from the cytoplasm to the lumen of the vacuole and also regulates cytosolic pH. V-ATPase is active in the plasma membrane of human tumor cells (11Martinez-Zaguilan R. Lynch R.M. Martinez G.M. Gillies R.J. Am. J. Physiol. 1993; 265: C1015-C1029Crossref PubMed Google Scholar), and V-ATPase genes are considered “housekeeping genes.” However, cytosolic pH is critical for the cytotoxicity of anticancer agents (12Laurencot C.M. Andrews P.A. Kennedy K.A. Oncol. Res. 1995; 7: 363-369PubMed Google Scholar), and cellular acidosis is thought to be a trigger for apoptosis and to play a role in drug resistance. Therefore, understanding the mechanisms regulating tumor acidity is important for developing new approaches to cancer chemotherapy. By using differential display, we have shown that one of the proton pump subunit genes, ATP6L (subunit c), is induced by cisplatin (13Murakami T. Sibuya I. Ise T. Zhe-Sheng C. Akiyama S. Nakagawa M. Izumi H. Nakamura T. Matsuo K. Yamada Y. Kohno K. Int. J. Cancer. 2001; 93: 869-874Crossref PubMed Scopus (123) Google Scholar), and several V-ATPase subunit genes are up-regulated in drug-resistant cell lines (13Murakami T. Sibuya I. Ise T. Zhe-Sheng C. Akiyama S. Nakagawa M. Izumi H. Nakamura T. Matsuo K. Yamada Y. Kohno K. Int. J. Cancer. 2001; 93: 869-874Crossref PubMed Scopus (123) Google Scholar, 14Martinez-Zaguilan R. Raghunand N. Lynch R.M. Bellamy W. Martinez G.M. Rojas B. Smith D. Dalton W.S. Gillies R.J. Biochem. Pharmacol. 1999; 57: 1037-1046Crossref PubMed Scopus (135) Google Scholar). Interaction of the V-ATPase c subunit with β1 integrin has been reported (15Skinner M.A. Wildeman A.G. J. Biol. Chem. 1999; 274: 23119-23127Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar,16Skinner M.A. Wildeman A.G. J. Biol. Chem. 2001; 276: 48451-48457Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), and β1 integrin-mediated signaling prevents lung cancer cells from drug-induced apoptosis. The level of the V-ATPase c subunit may be critical for V-ATPase activity. In order to study transcriptional regulation of the c subunit at the molecular level, we have identified its promoter sequences and characterized the transcription factors that regulate its expression in cancer cells. We hypothesized that V-ATPase expression is up-regulated in response to cellular acidosis and show that c subunit promoter activity is activated by treatment with anticancer agents, especially the DNA topoisomerase II inhibitor, TAS-103 (17Azuma R. Urakawa A. J. Chromatogr. B. Biomed. Appl. 1997; 691: 179-185Crossref Scopus (8) Google Scholar, 18Byl J.A. Fortune J.M. Burden D.A. Nitiss J.L. Utsugi T. Yamada Y. Osheroff N. Biochemistry. 1999; 38: 15573-15579Crossref PubMed Scopus (61) Google Scholar), which can induce cellular acidosis (19Kluza J. Lansiaux A. Wattez N. Mahieu C. Osheroff N. Bailly C. Cancer Res. 2000; 60: 4077-4084PubMed Google Scholar). We show also that the levels of two transcription factors, Sp1 and Oct1, increase in response to genotoxic stress and that V-ATPase inhibition strongly enhances TAS-103-induced apoptosis. ATP6Lgenomic clones were isolated from a human placental genomic library in EMBL3 by screening with cDNA. All positive phage were mapped with EcoRI and SalI. Several genomic fragments were also used as hybridization probes to confirm the overlapping regions. Two genomic DNA fragments around the first exon were subcloned into pUC18 (Fermentas AB, Lithuania) and sequenced with an Automated sequencer 377 (PE Applied Biosystems). The primer, 5′-GTCACATGACCTGGGCCCCG-3′, derived from the first exon ofATP6L, was labeled at its 5′ end and hybridized with poly(A) RNA from KB cells in 80% formamide, 0.4 m NaCl, 40 mm PIPES (pH 6.4), and 1 mm EDTA for 4 h at 52 °C. The primer-RNA hybrid was precipitated and resuspended in reverse transcriptase mixture (Invitrogen). After 1 h of incubation at 42 °C, the reaction was terminated by making the solution 20 mm in EDTA. The RNA was hydrolyzed with 0.125m NaOH for 1 h at 65 °C, the reaction neutralized, and the extended DNA then precipitated with alcohol. The DNA was analyzed on a 7 m urea, 6% polyacrylamide gel to determine the size of the extended product. Sequencing reactions using the same primer were similarly analyzed. Human epidermoid cancer KB cells (20Shen D.W. Akiyama S. Schoenlein P. Pastan I. Gottesman M.M. Br. J. Cancer. 1995; 71: 676-683Crossref PubMed Scopus (81) Google Scholar), human prostate cancer PC3 cells (21Nakagawa M. Nomura Y. Kohno K. Ono M. Mizoguchi H. Ogata J. Kuwano M. J. Urol. 1993; 150: 1970-1973Crossref PubMed Scopus (32) Google Scholar), and human breast cancer MCF7 cells (22Furuya Y. Yamamoto K. Kohno N., Ku, Y. Saitoh Y. Cancer Lett. 1994; 81: 95-98Crossref PubMed Scopus (19) Google Scholar) were cultured in Eagle's minimal essential medium (Nissui Seiyaku Co., Tokyo, Japan) or Dulbecco's modified Eagle medium (Nissui Seiyaku Co., Tokyo, Japan) containing 10% fetal bovine serum, 0.292 mg/ml l-glutamine, 100 units/ml penicillin, and 100 μg/ml kanamycin. The anti-Sp1 (catalogue number sc-420 for supershift assay, sc-59 for chromatin immunoprecipitation assay, and Western blotting), anti-Sp3 (sc-644), anti-Oct1 (sc-232), and anti-Oct2 (sc-233) antibodies were purchased from Santa Cruz Biotechnology. Antiserum to V-ATPase subunit E was generated by multiple immunization of a New Zealand White rabbit with synthetic peptides as described (23Miura K. Miyazaki S. Furuta S. Mitsushita J. Kamijo K. Ishida H. Miki T. Suzukawa K. Resau J. Copeland T.D. Kamata T. J. Biol. Chem. 2001; 276: 46276-46283Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The sequence of the synthetic peptides is ALFGANANRKFLD. Total RNA from KB cells was isolated using Sepasol reagent (Nacalai Tesque, Kyoto, Japan). RNA samples (20 μg/lane) were separated on a 1% formaldehyde-agarose gel and transferred to a Hybond N+ filter (Amersham Biosciences) with 10× SSC. Prehybridization and hybridization were performed as described (24Koike K. Abe T. Hisano T. Kubo T. Wada M. Kohno K. Kuwano M. Jpn. J. Cancer Res. 1996; 87: 765-772Crossref PubMed Scopus (39) Google Scholar). For analysis of stability of V-ATPase subunit transcripts by cisplatin or TAS-103, KB cells were treated with actinomycin D (1 μg/ml) and cisplatin (10 μm) or TAS-103 (4 μm) for 6 h. Cisplatin was purchased from Sigma, and TAS-103 was kindly provided from Taiho Pharmaceutical Co., Ltd. (Tokyo, Japan). KB cells were treated with or without TAS-103 for 12 h. Briefly, cells were homogenized in 0.25 m sucrose, and the homogenates were centrifuged at 3,000 rpm for 10 min. The supernatant was centrifuged at 15,000 rpm for 30 min. The pellets were resuspended to 0.25 m sucrose. The resuspension was overlaid with 2.10 and 1.25 m sucrose cushions and centrifuged at 24,000 rpm for 12 h. The membrane fractions at the 0.25–1.25 m sucrose interface were collected and used for Western blotting. For metabolic labeling, KB cells in a 100-mm tissue culture dish were cultured in Dulbecco's methionine and cysteine-free modified Eagle's medium (Invitrogen) supplemented with 1% dialyzed fetal calf serum and were labeled with 50 μCi/ml [35S]methionine and -cysteine labeling mixture (AmershamBiosciences) with or without 4 μm TAS-103 for 12 h. After washing the cells twice with ice-cold phosphate-buffered saline (PBS), cells were lysed in RIPA buffer (50 mm Tris-HCl (pH 7.5), 1 mm EDTA, 150 mm NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 1 mm PMSF). After centrifugation at 3,000 rpm for 5 min at 4 °C, 2 mg of supernatant (cellular fraction) were incubated with antiserum to V-ATPase subunit E or preimmune binding to 15 μl of protein A/G-agarose. The mixtures were incubated for 12 h at 4 °C and washed three times with RIPA buffer. Immunoprecipitation samples and 1% of preimmunoprecipitation samples (input) were simultaneously separated on a 15 or 10% SDS-PAGE and autoradiography. The EcoRI and NotI fragment (nt −1627 to nt +194) of the ATP6L gene into theSmaI site of basic vector 2 (Nippon Gene, Tokyo) was designed pV-ATPase c Luc1. For the construction of deletion constructs, it was digested with PstI (pV-ATPase c Luc2),BshTI (pV-ATPase c Luc6), and NarI (pV-ATPase c Luc7). The digestion products were self-ligated. Other constructs (pV-ATPase c Luc3, -4, and -5) were constructed by PCR (Fig.7 A). The pV-ATPase c Luc3m1, Luc3m2, Luc3m3, Luc3SR, and Luc3–5bp were constructed by PCR-based method using mutated oligonucleotides (Figs. 7 B and 9 B). ATP6Egenomic clones were isolated from a human placental genomic library, and the 5′-flanking region of the ATP6E(nt −715 to +132) gene was subcloned in basic vector 2 (pV-ATPase E Luc1). Sp1 cDNA (encoding amino acids 30 to C-terminal) was kindly provided by Dr. Robert Tjian (University of California, Berkeley), and theXhoI fragment added start codon at the N-terminal start codon was ligated in pcDNA3 vector (Amersham Biosciences). For expression plasmids, full-length cDNA fragments of human Sp3, Oct1, and Oct2 were generated by reverse transcription-PCR using total RNA from KB cells and cloned into the pcDNA3 vector. The following oligonucleotides were used for cDNA constructions: Sp3, 5′-ATGGCTGCCTTGGACGTGGATAGC-3′ and 5′-TTACTCCATTGTCTCATTTCCAGAAAC-3′; Oct1, 5′-ATGAACAATCCGTCAGAAACCAGTAAACC-3′ and 5′-TCACTGTGCCTTGGAGGCGGTGGTGG-3′; Oct2, 5′-ATGGTTCACTCCAGCATGGGGGC-3′ and 5′-TTACCCCGTGCTGGGGTTCAGG-3′.Figure 9Cooperation between Sp1 and Oct1 at theATP6Lpromoter. A, EMSA with mutant forms of Oligo3. Labeled oligonucleotides with reversed GC box (Oligo3SR) or a 5-bp sequence inserted between the Sp1 and Oct1 sites (Oligo3–5bp) were prepared as described under “Materials and Methods.” Buffer S nuclear extracts of KB cells treated with TAS-103 (4 μm) were incubated with probes and 2 μg of anti-Sp1 or anti-Oct1 antibody (Ab) for 30 min at 4 °C. The slower migrating band (C3) is a complex formed with both Sp1 and Oct1, the faster migrating band (C6) is formed with either Sp1 or Oct1. C1–6refer to the following: C1, Sp1 + Oct1 + anti-Oct1 antibody; C2, Sp1 + Oct1 + anti-Sp1 antibody;C3, Sp1 + Oct1; C4, Oct1 + anti-Oct1 antibody; C5, Sp1 + anti-Sp1 antibody;C6, Sp1 or Oct1. B, luciferase assay with the mutant reporter plasmids. Reporter plasmids with reversed GC box (Luc3SR), or a 5-bp insertion between the Sp1- and Oct1-binding site (Luc3–5bp), were constructed. The mutated sequences are 5′-GGCGGGCGTATGCTAAT-3′ (Luc3SR) and 5′-CCGCCCCGTctagaATGCTTTT-3′ (Luc3–5bp). Luciferase assay was carried out as described under “Materials and Methods.”View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cells were seeded into 12-well tissue culture plates at a concentration of 4 × 104KB cells, PC3 cells, and MCF7 cells. On the following day, cells were transfected with 0.4 μg of luciferase reporter plasmid DNA using 2 μl of Superfect reagent (Qiagen, Germany) according to the manufacturer's instructions. The β-galactosidase reporter gene (pSV-β-gal, Nippon Gene, Tokyo) was co-transfected as an internal control. After transfection for 12 h, the cells were washed, incubated at 37 °C for 12 h in fresh medium or in medium containing either TAS-103 (4 μm) or cisplatin (10 μm), and then harvested. For co-transfection experiments with Sp1, Sp3, Oct1, and Oct2 expression plasmids, PC3 cells were transfected with 0.2 μg of luciferase reporter plasmid (pV-ATPase c Luc3) and 0.4 μg of expression plasmid. After transfection for 12 h, the cells were incubated at 37 °C for 24 h in fresh medium and then harvested. Lysed cells were assayed for luciferase activity using a Picagene kit (Toyoinki, Tokyo, Japan); the light intensity was measured for 15 s with a luminometer (Dynatech ML1500, JEOL, Japan). The β-galactosidase enzyme assay was performed according to the protocol of Promega. KB cells and MCF7 cells were treated with dimethyl sulfate (DMS) in vivo (25Konishi T. Nomoto M. Shimizu K. Abe T. Itoh H. Friedrich H. Gunther E. Higashi K. J. Biochem. (Tokyo). 1995; 118: 1021-1029Crossref PubMed Scopus (24) Google Scholar). The extracted DNA was cleaved with 1 m piperidine at 90 °C for 30 min. As a control guanine ladder, naked genomic DNA from KB cells was reacted with DMS in vitro and cleaved with piperidine as described above. Ligation-mediated PCR was performed as described previously (25Konishi T. Nomoto M. Shimizu K. Abe T. Itoh H. Friedrich H. Gunther E. Higashi K. J. Biochem. (Tokyo). 1995; 118: 1021-1029Crossref PubMed Scopus (24) Google Scholar,26Nomoto M. Gonzalez F.J. Mita T. Inoue N. Kawamura M. Biochim. Biophys. Acta. 1995; 1264: 35-39Crossref PubMed Scopus (10) Google Scholar). The nucleotide sequences of individual primers are as follows: 5′-GCCTGCAGCTTCACGCC-3′ (−172 to −184) primer 1; 5′-CGCCGGGAACCCAACACCTGC-3′ (−135 to −115) primer 2; 5′-CCGGGAACCCAACACCTGCAGACGACGC-3′ (−133 to −106) primer 3 for analysis of the lower strand of the subunit c gene. Primers 1 and 2 were used for the first strand synthesis and PCR amplification, respectively. Primer 3 was labeled at the 5′ end with [γ-32P]ATP and used for final detection of the ladder. Protein-DNA cross-linking was performed by incubating KB cells with formaldehyde at a final concentration of 1% for 10 min at room temperature. Cells were washed with PBS and collected by centrifugation at 1,200 rpm for 5 min. Cells were then lysed in buffer X (50 mm Tris-HCl (pH 8.0), 1 mm EDTA, 120 mm NaCl, 0.5% Nonidet P-40, 10% glycerol, and 1 mm PMSF) for 15 min on ice. The lysate was sonicated with 10 pulses of 10 s each at 50–60% of maximum power with a sonicator (Taitec, Tokyo, Japan) equipped with a microtip to reduce the chromatin fragments to average sizes of less than 500 bp. Soluble chromatin was precleared by addition of 10 mg of protein A-Sepharose. An aliquot of precleared chromatin containing 1 × 106 cells was removed and used in the subsequent PCR analysis. The remainder of the chromatin was divided, with each having 1 × 106 cells, and diluted with buffer X. Then protein-DNA was incubated with 2 μg of anti-Sp1, anti-Oct1 antibody, and normal rabbit IgG in a final volume of 800 μl overnight at 4 °C. Immune complexes were collected by incubation with 15 μl of protein A/G-agarose for 1 h at 4 °C. Protein A/G-agarose pellets were washed once with 1 ml of buffer X, once with high salt buffer X (50 mm Tris-HCl (pH 8.0), 1 mm EDTA, 500 mm NaCl, 0.5% Nonidet P-40, 10% glycerol, and 1 mm PMSF), once with LiCl buffer (10 mm Tris, 1 mm EDTA, 0.25 m LiCl, 1% Nonidet P-40, and 1% sodium deoxycholate (pH 8.0)), and twice with TE (10 mmTris, 1 mm EDTA (pH 8.0)). Immune complexes were eluted twice with 250 μl of elution buffer (0.1 mNaHCO3, 1% SDS). To reverse the protein-DNA cross-linking, eluted samples were incubated with 0.2 m NaCl for 4 h at 65 °C. Samples were digested with proteinase K (0.04 mg/ml) for 2 h at 45 °C and then with RNase A (0.02 mg/ml) for 30 min at 37 °C. DNA was purified with phenol/chloroform followed by ethanol precipitation. Purified DNA was resuspended in 20 μl of H2O. Aliquots of 1 μl of serial dilution were analyzed by PCR with the appropriate primer pairs. The V-ATPase c promoter primers are as follows: 5′-CTGCAGACGACGCGCAGCCGCAGAGGAGGC-3′ and 5′-GCGCGAGACCGGTCCAACGCTGCGGAGATC-3′, and the YB-1promoter primers are 5′-AGATCTCTATCACGTGGCTGTTGC-3′ and 5′-AAGCTTATCAGTCCTCCATTCTCATTGG-3′. Amplification was performed for a pre-determined optimal number of cycles. PCR products were separated by electrophoresis on 2% agarose gel, which were stained with ethidium bromide. Nuclear extracts using buffer S of KB cells were prepared as described (27Rundlof A.K. Carlsten M. Arner E.S. J. Biol. Chem. 2001; 276: 30542-30551Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Briefly, 2 × 107 cells were collected with PBS, resuspended in 1 ml of ice-cold 10 mm HEPES-KOH (pH 7.9), 1.5 mmMgCl2, 10 mm KCl, 0.2 mm PMSF, 0.5 mm DTT, and incubated on ice for 15 min. The cells were lysed with a dropping of 0.6% Nonidet P-40, and the lysate was centrifuged at 3,000 rpm for 10 min. The resulting nuclear pellets were resuspended in 50 μl of ice-cold buffer S (20 mmHEPES-KOH (pH 7.9), 25% glycerol, 1.5 mmMgCl2, 10 mm KCl, 0.2 mm EDTA, 0.2 mm PMSF, and 0.5 mm DTT), and KCl was added to a final concentration of 0.4 m and incubated for 15 min on ice with frequent gentle mixing. Following centrifugation for 5 min at 4 °C in a microcentrifuge to remove insoluble material, the supernatant (nuclear extract) was stored at −70 °C. Nuclear extracts using buffer C were also prepared as described (28Ise T. Nagatani G. Imanura T. Kato K. Takano H. Nomoto M. Izumi H. Ohmori H. Okamoto T. Ohga T. Uchiumi T. Kuwano M. Kohno K. Cancer Res. 1999; 59: 342-346PubMed Google Scholar). Briefly, 2 × 107 cells were collected with PBS, resuspended in 1 ml of ice-cold 10 mm HEPES-KOH (pH 7.9), 10 mm KCl, 0.5 mm PMSF, 1 mm DTT, 0.1 mm EDTA, 0.1 mm EGTA, and incubated on ice for 15 min. The cells were lysed with a dropping of 0.6% Nonidet P-40, and the lysate was centrifuged at 3,000 rpm for 10 min. The resulting nuclear pellets were resuspended in 50 μl of ice-cold buffer C (20 mm HEPES-KOH (pH 7.9), 0.4 m NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm PMSF, and 1 mm DTT) and incubated for 15 min on ice with frequent gentle mixing. Following centrifugation for 5 min at 4 °C in a microcentrifuge to remove insoluble material, the supernatant was stored at −70 °C. Its protein concentration was determined by the method of Bradford. EMSAs were performed as described (27Rundlof A.K. Carlsten M. Arner E.S. J. Biol. Chem. 2001; 276: 30542-30551Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Briefly, 4 μg of nuclear extract proteins prepared with buffer S were incubated for 30 min at room temperature in a final volume of 20 μl containing 20 mm HEPES (pH 7.9), 1.5 mm MgCl2, 0.2 mm EDTA, 0.1 mm PMSF, 1 mm DTT, 7.5% glycerol, 0.5 μg of poly(dI-dC), and 1 × 104cpm (1 ng) of 32P-labeled oligonucleotide probe in the absence or presence of various competitors. On the other hand, 4 μg of nuclear extract proteins prepared with buffer C were incubated for 30 min at room temperature in a final volume of 20 μl containing 10 mm HEPES (pH 7.9), 50 mm NaCl, 1 mmMgCl2, 1 mm EDTA, 1 mm DTT, 8% glycerol, 0.1 μg of poly(dI-dC), and 1 × 104 cpm (1 ng) of 32P-labeled oligonucleotide probe. Products were analyzed on nondenaturing 4% polyacrylamide gels using a bioimaging analyzer (BAS 2000; Fuji Photo Film, Tokyo). The sequences of oligonucleotides used for EMSAs are as follows: Oligo1 (−118 to −89), 5′-CTGCAGACGACGCGCAGCCGCAGAGGAGGC-3′ and 3′-ACGTCTGCTGCGCGTCGGCGTCTCCTCCGC-5′; Oligo2 (−98 to −69), 5′-CAGAGGAGGCGGGGCGTCCGAGGCCCCGCC-3′ and 3′-TCTCCTCCGCCCCGCAGGCTCCGGGGCGGG-5′; Oligo3 (−78 to −49), 5′-AGGCCCCGCCCCGTATGCTAATGAAGCACA-3′ and 3′-CCGGGGCGGGGCATACGATTACTTCGTGTG-5′; Oligo4 (−58 to −29), 5′-ATGAAGCACACACCACACCGCCCCGCCCCG-3′ and 3′-ACTTCGTGTGTGGTGTGGCGGGGCGGGGCC-5′; Oligo5 (−38 to −9), 5′-CCCCGCCCCGGCGCGAGACCGGTCCAACGC-3′ and 3′-GGGCGGGGCCGCGCTCTGGCCAGGTTGCGA-5′; Oligo6 (−28 to +2), 5′-GCGCGAGACCGGTCCAACGCTGCGGAGATC-3′ and 3′-GCGCTCTGGCCAGGTTGCGACGCCTCTAGG-5′; Oligo3m1, 5′-AGGCCCTTCCCCGTATGCTAATGAAGCACA-3′ and 3′-CCGGGAAGGGGCATACGATTACTTCGTGTG-5′; Oligo3m2, 5′-AGGCCCCGCCCCGTATGCTTTTGAAGCACA-3′ and 3′-CCGGGGCGGGGCATACGAAAACTTCGTGTG-5′; Oligo3m3, 5′-AGGCCCTTCCCCGTATGCTTTTGAAGCACA-3′ and 3′-CCGGGAAGGGGCATACGAAAACTTCGTGTG-5′; Oligo3SR, 5′-AGGGGGGCGGGGGTATGCTAATGAAGCACA-3′ and 3′-CCCCCCGCCCCCATACGATTACTTCGTGTG-5′; Oligo3–5bp, 5′-AGGCCCCGCCCCGTCTAGAATGCTAATGAAGCACA-3′ and 3′-CCGGGGCGGGGCAGATCTTACGATTACTTCGTGTG-5′. For supershift assay, nuclear extracts were incubated with probes and 2 μg of anti-Sp1, anti-Sp3, anti-Oct1, and anti-Oct2 antibody for 30 min at 4 °C. Preparation of nuclear extracts and separation of membrane fractions were described above. Nuclear extracts (100 μg of protein) of KB cells prepared with buffer C were separated on a 10% SDS-PAGE, and membrane fractions (40 μg) by sucrose gradient of KB cells were separated on a 15% SDS-PAGE gel and transferred to polyvinylidene difluoride membrane (Millipore) using a semidry blotter. Prestained protein marker (Nacalai Tesque, Kyoto, Japan) was used as a molecular weight standard. Immunoblot analysis was performed with an appropriate dilution of each antibody. KB cells in a 100-mm tissue culture dish were treated with TAS-103, bafilomycin A1 (Wako, Ohsaka, Japan), and a combination of these drugs for 36 h. The cells were washed twice with ice-cold PBS and then collected by centrifugation at 1,500 rpm for 10 min. The cell pellets were resuspended in 500 μl of Tris-EDTA buffer (20 mm Tris-HCl (pH 8.0), 20 mm EDTA) containing 0.1% SDS and proteinase K (0.5 mg/ml) at 50 °C for 2 h and then with RNase A (0.02 mg/ml) for 30 min at 37 °C. DNA was purified with phenol/chloroform followed by ethanol precipitation. Purified DNA was resuspended in 100 μl of H2O. DNA samples were separated by electrophoresis on 2% agarose gel, which were stained with ethidium bromide (19Kluza J. Lansiaux A. Wattez N. Mahieu C. Osheroff N. Bailly C. Cancer Res. 2000; 60: 4077-4084PubMed Google Scholar). Cells were seeded into 12-well tissue culture plates at a concentration of 4 × 104 KB cells and treated with TAS-103, bafilomycin A1, and a combination of these drugs for 36 h. The cells were removed by trypsinization and resuspended in hypotonic cell lysis buffer (25 mm HEPES-KOH (pH 7.5), 5 mm MgCl2, 5 mm EDTA, 5 mm DTT, 2 mm PMSF, 10 μg/ml pepstatin A, and 10 μg/ml leupeptin). Following centrifugation for 20 min at 4 °C in a microcentrifuge, the supernatant fractions were collected. The fluorescence (CPP32 activity) of each sample was analyzed with the Fluorometric CaspACETMAssay System (Promega, Madison, WI) according to the manufacturer's instructions. For caspase activity with CaspACETMFITC-VAD-FMK in situ marker (Promega, Madison, WI), KB cells were treated with TAS-103, bafilomycin A1, and a combination of these drugs for 48 h. Cells were stained with CaspACETMFITC-VAD-FMK in situ marker according the manufacturer's instructions and analyzed using fluorescence microscopy. For statistical analysis of each experiment, 4 fields (×400) were counted per stimulation and cell type (between 300 and 400 cells in total). To isolate genomic clones encoding the 5′ region of theATP6L gene, a human genomic library was screened with a previously isolated ATP6LcDNA clone (13Murakami T. Sibuya I. Ise T. Zhe-Sheng C. Akiyama S. Nakagawa M. Izumi H. Nakamura T. Matsuo K. Yamada Y. Kohno K. Int. J. Cancer. 2001; 93: 869-874Crossref PubMed Scopus (123) Google Scholar). Two clones containing non-identical inserts were characterized. The restriction map of these clones is shown in Fig.1 A, and sequence analysis confirmed that they encode ATP6L. To localize the first exon more accurately, the promoter proximal plasmid was digested with restriction enzymes and analyzed by Southern blotting using cDNA. In order to determine the nucleotide sequence of the promoter region, a 1.9-kb EcoRI-SalI fragment of EMBL3 was subcloned into pUC18 (Fig. 1 A), and the nucleotide sequence of the first exon and its 5′-flanking region were determined (Fig.1 B). This fragment contained exons with sequences identical to the 5′ portion previously determined from cDNA. To define precisely the transcription initiation site, we performed primer extension. The cDNA products extended from the primer were analyzed by electrophoresis and sequenced using the same primer. Two major transcription initiation sites were observed (Fig.2). About 20% of the transcripts initiated at +1 and 80% initiated at +75. The transcription initiation site of the human gene is located 236 bp upstream from that of the mouse gene (29Wang S.P. Krits I. Bai S. Lee B.S. J. Biol. Chem. 2002; 277: 8827-8834Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). An additional 100-bp sequence of the 5′-untranslated region has been published. This indicates" @default.
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• W2169071938 title "Enhanced Expression of the Human Vacuolar H+-ATPase c subunit Gene (ATP6L) in Response to Anticancer Agents" @default.
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