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• W2015120197 abstract "Metallothionein I can be induced in response to a variety of agents that include heavy metals and oxidative stress. On the contrary, its induction was suppressed in some lymphoid-derived cancer cells. The mechanism of this repression has not been elucidated. Here, we show silencing of MT-I gene in a solid transplanted rat tumor as a result of promoter methylation at all the 21 CpG dinucleotides that span the region from −225 bp to +1 bp. By contrast, none of these CpG dinucleotides were methylated in the livers from the rats bearing the tumor, which was consistent with the efficient induction of the gene in this tissue by zinc sulfate. Genomic footprinting revealed lack of access of the transcriptional activators to the respective cis-acting elements of the methylatedMT-I promoter in the hepatoma. The absence of footprinting was not due to inactivation of the metal regulatory transcription factor MTF-1, because it was highly active in the hepatoma. Treatment of the hepatoma bearing rats with 5-azacytidine, a demethylating agent, induced basal as well as heavy metal-activated MT-I gene expression in the hepatoma, implying that methylation was indeed responsible for silencing the gene. Bisulfite genomic sequencing showed significant (>90%) demethylation of CpG dinucleotides spanningMT-I promoter in the hepatoma following treatment with 5-AzaC. The hypermethylation of MT-I promoter was probably caused by significantly higher (as much as 7-fold) level of DNA methyl transferase activity as well as enhanced expression of its gene in the hepatoma relative to the host liver. These data elucidated for the first time the molecular mechanism for the silencing of a highly inducible gene in a solid tumor transplanted in an animal, as compared with the robust induction in the corresponding parental tissue and have discussed the probable reasons for the suppression of this gene in some tumors. Metallothionein I can be induced in response to a variety of agents that include heavy metals and oxidative stress. On the contrary, its induction was suppressed in some lymphoid-derived cancer cells. The mechanism of this repression has not been elucidated. Here, we show silencing of MT-I gene in a solid transplanted rat tumor as a result of promoter methylation at all the 21 CpG dinucleotides that span the region from −225 bp to +1 bp. By contrast, none of these CpG dinucleotides were methylated in the livers from the rats bearing the tumor, which was consistent with the efficient induction of the gene in this tissue by zinc sulfate. Genomic footprinting revealed lack of access of the transcriptional activators to the respective cis-acting elements of the methylatedMT-I promoter in the hepatoma. The absence of footprinting was not due to inactivation of the metal regulatory transcription factor MTF-1, because it was highly active in the hepatoma. Treatment of the hepatoma bearing rats with 5-azacytidine, a demethylating agent, induced basal as well as heavy metal-activated MT-I gene expression in the hepatoma, implying that methylation was indeed responsible for silencing the gene. Bisulfite genomic sequencing showed significant (>90%) demethylation of CpG dinucleotides spanningMT-I promoter in the hepatoma following treatment with 5-AzaC. The hypermethylation of MT-I promoter was probably caused by significantly higher (as much as 7-fold) level of DNA methyl transferase activity as well as enhanced expression of its gene in the hepatoma relative to the host liver. These data elucidated for the first time the molecular mechanism for the silencing of a highly inducible gene in a solid tumor transplanted in an animal, as compared with the robust induction in the corresponding parental tissue and have discussed the probable reasons for the suppression of this gene in some tumors. 5-azacytidine DNA methyl transferase metallothionein electrophoretic mobility shift assay glyceraldehyde-3-phosphate dehydrogenase polymerase chain reaction base pair(s) metal regulatory element ligation-mediated Methylation at the 5 position of cytosine is a unique modification of eukaryotic genome that occurs within CpG dinucleotides of some genes (for recent reviews see Refs. 1Baylin S.B. Herman J.G. Graff J.R. Vertino P.M. Issa J.P. Adv. Cancer Res. 1998; 72: 141-196Crossref PubMed Google Scholar, 2Tajima S. Suetake I. J. Biochem. (Tokyo). 1998; 123: 993-999Crossref PubMed Scopus (60) Google Scholar, 3Jones P. Nat. Genet. 1999; 21: 163-167Crossref PubMed Scopus (2040) Google Scholar). This DNA modification has evolved as an epigenetic mechanism in vertebrates, plants, and fungi but not in yeast, flies, and nematodes. Although CpG dinucleotides are present within the coding region, clusters of this sequence known as CpG islands occur frequently in the promoter regions of some genes. The CpG regions are usually devoid of methylation, but in some cancer cells, these sites appear to be susceptible to methylation. In normal cells, only a few genes on the inactive X chromosome and foreign DNA,e.g. transposable elements and provirus are silenced by this mechanism (4Li E. Beard C. Forster A.C. Bestor T.H. Jaenisch R. Cold Spring Harbor Symp. Quant. Biol. 1993; 58: 297-305Crossref PubMed Scopus (85) Google Scholar, 5Jaenisch R. Beard C. Lee J. Marahrens Y. Panning B. Novartis Found. Symp. 1998; 214: 200-232PubMed Google Scholar, 6Walsh C.P. Bestor T.H. Genes Dev. 1998; 13: 26-34Crossref Scopus (348) Google Scholar). Many cancer cells have been shown to exhibit global hypomethylation of DNA compared with control cells (7Feinberg A.P. Gehrke C.W. Kuo K.C. Ehrlich M. ‘ Res. 1988; 48: 1159-1161Google Scholar, 8Gama-Sosa M.A. Slagel V.A. Trewyn R.W. Oxenhandler R. Kuo K.C. Gehrke C.W. Ehrlich M. Nucleic Acids Res. 1983; 11: 6883-6894Crossref PubMed Scopus (688) Google Scholar), which is consistent with the oncogenic potential of cells following treatment with 5-azacytidine (5-AzaC),1 a demethylating agent (9Santi D.V. Garrett C.E. Barr P.J. Cell. 1983; 33: 9-10Abstract Full Text PDF PubMed Scopus (340) Google Scholar). On the contrary, the promoters of some tumor suppressor genes are methylated, resulting in their inactivation (1Baylin S.B. Herman J.G. Graff J.R. Vertino P.M. Issa J.P. Adv. Cancer Res. 1998; 72: 141-196Crossref PubMed Google Scholar). Mutation in the coding region of the tumor suppressor genes has been generally considered the major mechanism of inactivation of the tumor suppressor genes. Recently, aberrant DNA methylation of the CpG islands in the promoter region has emerged as an alternative mechanism for the silencing of these growth regulatory genes and may be one of the earliest events in the neoplastic transformation of cells (10Toyota M. Ho C. Ahuja N. Ohe-Toyota M. Herman J.G. Baylin S.B. Issa J.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8681-8686Crossref PubMed Scopus (2089) Google Scholar). Indeed, silencing of the tumor suppressor genes (e.g. p16, RB, e-cad, ER, VHL, APC, p53,WT1, and BRCA1) by promoter methylation is known to occur in many tumor types (1Baylin S.B. Herman J.G. Graff J.R. Vertino P.M. Issa J.P. Adv. Cancer Res. 1998; 72: 141-196Crossref PubMed Google Scholar, 10Toyota M. Ho C. Ahuja N. Ohe-Toyota M. Herman J.G. Baylin S.B. Issa J.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8681-8686Crossref PubMed Scopus (2089) Google Scholar, 11Pogribny I.P. Miller B.J. James S.J. Cancer Lett. 1997; 115: 31-38Crossref PubMed Scopus (127) Google Scholar, 12Hiltunen M.O. Koistinaho J. Alhonen L. Myohanen S. Marin S. Kosma V.M. Paakkonen M. Janne J. Br. J. Cancer. 1997; 76: 1124-1130Crossref PubMed Scopus (38) Google Scholar, 13Schroeder M. Mass M.J. Biochem. Biophys. Res. Commun. 1997; 235: 403-406Crossref PubMed Scopus (74) Google Scholar, 14Mancini D.N. Rodenhiser D.I. Ainsworth P.J. O'Malley F.P. Singh S.M. Xing W. Archer T.K. Oncogene. 1998; 16: 1161-1169Crossref PubMed Scopus (176) Google Scholar). Promoter methylation is also responsible for some genetic diseases, e.g. fragile X, Prader-Wille, and Angelman syndromes (15Schwemmle S. de Graaff E. Deissler H. Glaser D. Wohrle D. Kennerknecht I. Just W. Oostra B.A. Dorfler W. Vogel W. Steinbach P. Am. J. Hum. Genet. 1997; 60: 1354-1362Abstract Full Text PDF PubMed Scopus (46) Google Scholar, 16Kosaki K. McGinniss M.J. Veraksa A.N. McGinnis W.J. Jones K.L. Am. J. Med. Genet. 1997; 73: 308-313Crossref PubMed Google Scholar). In recent years, there has been growing interest in the elucidation of the molecular mechanisms by which DNA methylation represses gene expression. Recent studies have identified two repressor proteins, MeCP1 and MeCP2, that bind specifically to methyl-CpG without apparent sequence specificity and repress methylated promoter activity in vitro as well as in vivo (17Meehan R.R. Lewis J.D. McKay S. Kleiner E.L. Bird A.P. Cell. 1989; 58: 499-507Abstract Full Text PDF PubMed Scopus (519) Google Scholar, 18Lewis J.D. Meehan R.R. Henzel W.J. Maurer-Fogy I. Jeppesen P. Klein F. Bird A. Cell. 1992; 69: 905-914Abstract Full Text PDF PubMed Scopus (1062) Google Scholar, 19Nan X. Cross S. Bird A. Novartis Found. Symp. 1998; 214: 6-50PubMed Google Scholar). Methylated genes can be transcribed efficiently in vitro in the absence of MeCPs. This finding suggests that CpG methylation does not by itself render these sites inaccessible to the basal transcriptional machinery or prevent interaction of the transcription factors with the promoters. Our laboratory has been involved in studying the molecular mechanisms for the control of metallothionein (MT) expression (20Aniskovitch L.P. Jacob S.T. Arch. Biochem. Biophys. 1997; 341: 337-346Crossref PubMed Scopus (15) Google Scholar, 21Aniskovitch L.P. Jacob S.T. Oncogene. 1998; 16: 1475-1486Crossref PubMed Scopus (12) Google Scholar, 22Datta P.K. Jacob S.T. Biochem. Biophys. Res. Commun. 1997; 230: 159-163Crossref PubMed Scopus (19) Google Scholar, 23Ghoshal K. Wang Y. Sheridan J.F. Jacob S.T. J. Biol. Chem. 1998; 273: 27904-27910Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 24Ghoshal K. Li Z. Jacob S.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10390-10395Crossref PubMed Scopus (17) Google Scholar). The rodents express four isoforms of MT, designated MT-I, MT-II, MT-III, and MT-IV. The first two isoforms are expressed in all tissues, whereas MT-III and MT-IV are expressed almost exclusively in the brain and the stratified epithelium of skin, tongue, etc., respectively (25Aschner M. Cherian M.G. Klaassen C.D. Palmiter R.D. Erickson J.C. Bush A.I. Toxicol. Appl. Pharmacol. 1997; 142: 229-242Crossref PubMed Scopus (179) Google Scholar, 26Quaife C.J. Findley S.D. Erickson J.C. Froelic G.J. Kelly E.J. Zambrowicz B.P. Palmiter R.D. Biochemistry. 1994; 33: 7250-7259Crossref PubMed Scopus (487) Google Scholar). The constitutive levels of MT-I and MT-II in most tissues are negligible but can be induced rather dramatically by a variety of agents that include heavy toxic metals, UV radiation, restraint stress, and agents that produce reactive oxygen species (for a review see Refs. 27Kagi J.A. Methods Enzymol. 1991; 205: 613-626Crossref PubMed Scopus (733) Google Scholar, 28Palmiter R.D. Experientia. 1987; 52 (suppl.): 63-80Google Scholar, 29Thiele D.J. Nucleic Acids Res. 1992; 20: 1183-1191Crossref PubMed Scopus (243) Google Scholar). Overexpression of MT in cells can confer resistance to some of these agents (30Kelley S.L. Basu A. Teicher B.A. Hacker M.P. Hamer D.H. Lazo J.S. Science. 1988; 241: 1813-1815Crossref PubMed Scopus (605) Google Scholar, 31Kang Y.J. Chen Y. Yu A. Voss-McCowan M. Epstein P.N. J. Clin. Invest. 1997; 100: 1501-1506Crossref PubMed Scopus (227) Google Scholar, 32Kang Y.J. Li G. Saari J.T. Am. J. Physiol. 1999; 276: H993-H997PubMed Google Scholar), whereas disruption of the MT genes renders the cells/tissues significantly more sensitive to these (33Masters B.A. Kelly E.J. Quaife C.J. Brinster R.L. Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 584-588Crossref PubMed Scopus (562) Google Scholar, 34Lazo J.S. Kondo Y. Dellapiazza D. Michalska A.E. Choo K.H. Pitt B.R. J. Biol. Chem. 1995; 270: 5506-5510Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 35Kondo Y. Woo E.S. Michalska A.E. Choo K.H. Lazo J.S. Cancer Res. 1995; 55: 2021-2023PubMed Google Scholar). The latter studies indicate a protective role for metallothionein against cellular damage and prompted us to explore the molecular mechanisms for MT expression. Although heavy metals or other inducers up-regulate the expression ofMT-I and MT-II genes in most tissues or cells in culture, there are a few exceptions. For example, none of these genes are induced in the lymphoid-derived tumor cells W7 and S49 by agents like heavy metals, glucocorticoids, etc. (36Compere S.J. Palmiter R.D. Cell. 1981; 25: 233-240Abstract Full Text PDF PubMed Scopus (274) Google Scholar). The molecular mechanism(s) for the suppression of MT induction in these cells has not been elucidated. Further, the status of MT expression in the parental cells (e.g. thymus) has not been explored. It is also unknown whether repression of MT induction is unique to cancer cells of lymphoid origin and whether such repression is observed in solid tumors growing in animals, which are the prevalent forms of human cancer. The present study was undertaken to test this possibility. For this purpose, we studied the expression of MT-I in a rat hepatoma in response to heavy metals and compared it with that in the liver of the tumor-bearing rats. The data show that the hepatoma is incapable of expressing MT-I following treatment of the hepatoma-bearing rats with CdSO4 or ZnSO4, whereas it is induced in the host liver. Further, this investigation has elucidated the mechanism for the repression of MT gene expression in the tumor. Morris hepatoma 3924A is a poorly differentiated, rapidly growing tumor with a mean doubling time of 4–5 days (37Duceman B.W. Rose K.M. Jacob S.T. J. Biol. Chem. 1981; 256: 10755-10758Abstract Full Text PDF PubMed Google Scholar). It is grown by transplanting a thin slice (0.5 × 2–3 mm) of the solid tumor with a trocher in the hind leg of rats (ACI strain). The tumor on each leg grows to 15–20 g within 4–5 weeks. Most experiments were performed when the tumors attained this size. For heavy metal treatment, the tumor-bearing rats were injected intraperitoneally with ZnSO4 (200 μmol/kg body weight), CdSO4 (15 μmol/kg body weight), or the same volume of physiological saline. After 4 h, the animals were sacrificed, a fraction of the liver, and the hepatoma were frozen in liquid N2 for RNA isolation, and the remaining tissues were processed for the isolation of nuclei. For 5-AzaC treatment, rats bearing the hepatoma were injected intraperitoneally on alternate day with the drug intraperitoneally (5 mg/kg body weight) dissolved in physiological saline or with saline alone (control) after 18 days of tumor pass (when tumor growth was obvious). After 2 weeks of treatment the rats were injected intraperitoneally with either ZnSO4 (200 μmol/kg) or saline and sacrificed after 4 h to isolate the liver and hepatoma DNA and RNA. Saline injected animals were used as control. No visible adverse effect of the drug on the animals was observe during this period of treatment, but the tumor growth was inhibited significantly. Total RNA was isolated from the liver and the hepatoma by guanidinium thiocyanate-acid phenol method (38Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62909) Google Scholar). 30 μg of RNA was separated by formaldehyde-agarose (1.2%) gel electrophoresis and transferred to nylon membrane. The membrane was then hybridized to mouse random-primed, [α-32P]dCTP-labeled MT-I minigene (39Glanville N. Durnam D.M. Palmiter R.D. Nature. 1981; 292: 267-269Crossref PubMed Scopus (182) Google Scholar), mouse DNA methyltransferase (DNA-MTase)-1 (40Bestor T. Laudano A. Mattaliano R. Ingram V. J. Mol. Biol. 1988; 203: 971-983Crossref PubMed Scopus (700) Google Scholar), or rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (41Piechaczyk M. Blanchard J. M. Marty L. Dani C. Panabieres F. ElSabouty S. Fort P. Jeanteur P. Nucleic Acids Res. 1984; 12: 6951-6963Crossref PubMed Scopus (406) Google Scholar) cDNAs as probes in rapid hybridization buffer (Amersham Pharmacia Biotech) following the manufacturer's protocol. The liver and the hepatoma nuclei were isolated by homogenization in high density sucrose buffer following the protocol of Gorski et al. (42Gorski K. Carneiro M. Schibler U. Cell. 1986; 47: 767-776Abstract Full Text PDF PubMed Scopus (969) Google Scholar) and Rose et al. (43Rose K.M. Stetler D.A. Jacob S.T. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 2833-2837Crossref PubMed Scopus (72) Google Scholar), respectively. The nuclei were then used for in vivofootprinting to make nuclear extract for assay of DNA-binding proteins and for DNA-MTase assay. The DNA-binding proteins were extracted with high salt (0.3m KCl) from the isolated nuclei following the protocol of Wadzinski et al. (44Wadzinski B.E. Wheat W.H. Jaspers S. Peruski Jr., L.F. Lickteig R.L. Johnson G.L. Klemm D.J. Mol. Cell. Biol. 1993; 13: 2822-2834Crossref PubMed Scopus (280) Google Scholar). Protein in the extract was estimated according to Bradford's method with bovine serum albumin as standard using Bio-Rad reagent. The DNA binding activities of MTF-1 and Sp 1 were measured by EMSA using specific oligonucleotide probes and 10 μg of the extract. These reagents were incubated with 0.1–0.5 ng of the32P-labeled MRE-d oligo in the buffer containing 10 mm Hepes (pH 7.9), 60 mm KCl, 5 mmMgCl2, 0.5 mm dithiothreitol, 10% glycerol, and 2 μg of poly(dI-dC) (23Ghoshal K. Wang Y. Sheridan J.F. Jacob S.T. J. Biol. Chem. 1998; 273: 27904-27910Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 45Radtke F. Heuchel R. Georgiev O. Hergersberg M. Gariglio M. Dembic Z. Schaffner W. EMBO J. 1993; 12: 1355-1362Crossref PubMed Scopus (329) Google Scholar). For competition, the extract was preincubated with 100-fold molar excess of the unlabeled competitor oligonucleotides (Sp 1, MRE-s) for 15 min on ice before addition of the labeled oligo. The reaction mixture was incubated on ice for 30 min, and the DNA-protein complexes were resolved by polyacrylamide gel electrophoresis (4% acrylamide; acrylamide:bisacrylamide = 38.7:1.3) in 0.25 × TBE running buffer. For antibody supershift assay, the reaction mixture at the end of the reaction was incubated with 1 μl of anti-MTF-1 antisera, a generous gift from Dr. Walter Schaffner, or antibodies against p70 subunit of Ku protein (Santa Cruz Biotechnology). The sequences of the upper strand of the deoxyoligonucleotides used in the synthesis of probe for EMSA are as follows: (a) MRE-d oligo (for Sp 1 and MTF-1) 5′-GATCCAGGGAGCTCTGCACTCCGCCCGAAAAGTA-3′; (b) MRE-s oligo (for MTF-1) 5′-GATCCAGGGAGCTCTGCACaCgGCCCGAAAAGTA-3′ (the letters in lowercase denote the mutations that abolished Sp 1 binding); and (c) Sp 1 5′-ATTCGATCGGGGCGGGGCGAGC-3′. Nuclei were isolated by sucrose density gradient centrifugation of both hepatoma and liver from the hepatoma bearing rats as described in the earlier section. In vivo methylation of nuclear DNA by dimethyl sulfate and the subsequent DNA preparation were performed following the protocol of Bossard et al. (46Bossard P. McPherson C.E. Zaret K.S. Methods. 1997; 11: 180-188Crossref PubMed Scopus (24) Google Scholar). For the preparation of control genomic G ladder, DNA was isolated from both liver and hepatoma nuclei followed by dimethyl sulfate treatment. Ligation-mediated PCR was carried out according to the procedure of Mueller and Wold (47Mueller P.R. Wold B. Science. 1989; 246: 780-786Crossref PubMed Scopus (787) Google Scholar), with modifications as described by Ping et al. (48Ping D. Jones P.L. Boss J.M. Immunity. 1996; 4: 455-469Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The MRE elements and MLTF/ARE site were analyzed using one set of upper strand and one set of lower strand specific primers. Sequences of the primers used to read the lower and the upper strand, and the conditions for LM-PCR are described by Majumder et al. (49Majumder S. Ghoshal K. Li Z. Jacob S.T. J. Biol. Chem. 1999; 274: 28584-28589Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Genomic DNA from the hepatoma and the liver from rats bearing Morris hepatoma 3924A was isolated. DNA (5 μg) was denatured in 0.3 mNaOH for 30 min at 37 °C in 10 μl of volume, mixed with 100 μl of 2 m sodium metabisulphite (Sigma) containing 0.5 mm hydroquinone (pH 5.0) and cycled in a thermal cycler at 50 °C for 30 min and 95 °C for 2 min for 20 cycles. The bisulfite-treated DNA was then desalted using Wizard DNA Clean-up Kit (Promega) and eluted in 100 μl of H2O. The DNA was then desulfonated in the presence of 0.3 m NaOH at 37 °C for 30 min. The solution was neutralized by addition of NaOAc (pH 4.5) to 0.2 m (final concentration). The bisulfite-converted DNA was then desalted again as stated before and eluted in 70 μl of H2O, and an aliquot (0.5–1 μl) was used for each PCR amplification. The metallothionein promoter from −304 to +148 bp was amplified using two sets of primers from the bisulfite-treated liver and hepatoma DNA. The PCR protocol used is as follows: 50 μl of reaction mixture contains 200 μm dNTPs, 2 μm primers, 2 mm MgCl2, 50 mm KCl, 20 mm Tris-HCl (pH 8.4), and 2.5 units of Taq DNA polymerase (Life Technologies, Inc.). Hot start amplification was performed in a Perkin-Elmer Thermal Cycler under the following conditions: 94 °C/2 min × 1 cycle, 94 °C/2 min, 60 °C/1 min, 72 °C/2 min × 35 cycles; and 72 °C/10 min × 1 cycle. To avoid any nonspecific amplification, product from the first round of PCR was then subjected to amplification with a set of nested primers under the same PCR condition, with the exception that the annealing temperature was maintained at 59 °C. Sequences of the nested primers used to amplify the upper strand of rat MT-Ipromoter from the bisulfite-treated DNA and the annealing temperatures are described earlier (49Majumder S. Ghoshal K. Li Z. Jacob S.T. J. Biol. Chem. 1999; 274: 28584-28589Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). To test the efficiency of bisulfite reaction, the amplified DNA (452 bp) was first digested with the restriction enzymes ApoI (R↓ AATT↑Y) orTsp509I (↓AATT↑) that can cut only the converted DNA but not the unconverted DNA. The restriction sites for these two enzymes are generated only when C residues are converted to T residues. The efficiency of bisulfite conversion was confirmed by complete restriction cut of the amplified DNA with Tsp509I orApoI. Amplified DNA was purified by agarose gel electrophoresis, followed by cleaning the eluted DNA using Wizard DNA Clean-up kit. The PCR product was directly sequenced using fmol sequencing kit (Promega) with the S2 and A2 primers (49Majumder S. Ghoshal K. Li Z. Jacob S.T. J. Biol. Chem. 1999; 274: 28584-28589Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The nuclei isolated from rat liver and hepatoma tissue were resuspended in 5 volumes of the following buffer: 50 mmTris-HCl (pH 7.8), 1 mm EDTA, 1 mmdithiothreitol, 0.01% sodium azide, 0.2 mmphenylmethylsulfonyl fluoride, 10% glycerol, 1% Tween 80 and lysed as described by Fiala et al. (50Fiala E.S. Staretz M.E. Pandya G.A. El-Bayoumy K. Hamilton S.R. Carcinogenesis. 1998; 19: 597-604Crossref PubMed Scopus (117) Google Scholar). Briefly, the method consists of passing the nuclear suspension through a 25 gauge needle, freezing on dry ice, and subsequent thawing at 37 °C, with the freeze-thaw cycle repeated three times. The suspension was centrifuged at 800 × g for 5 min at 4 °C. The protein content was determined using Bio-Rad Protein Assay reagent with bovine serum albumin as the standard. The activity of DNA-MTase was determined essentially as described by Tollefsbol and Hutchison (51Tollefsbol T.O. Hutchison C.A.r. J. Mol. Biol. 1997; 269: 494-504Crossref PubMed Scopus (51) Google Scholar). To compare the inducibility of MT-I in response to heavy metals in the liver and the hepatoma, the hepatoma-bearing rats were injected with zinc and cadmium salts, and MT-I mRNA level was measured 4 h post injection. Unexpectedly, the tumor was not responsive to either of the metals, whereas the liver expressed MT-I under these conditions (Fig. 1, compare lanes 1–3 withlanes 4–6, respectively). There was no detectable MT-I mRNA induction in the hepatoma even after 8 h and 24 h of zinc and cadmium treatment, respectively (data not shown). The MT-I induction in the liver was 10- and 20-fold after zinc and cadmium treatment, respectively. The reason for the relatively smaller extent of MT induction in this study is probably due to the higher basal level of MT mRNA in the host liver of the tumor bearing animals relative to the livers of normal ones (52Kloth D.M. Chin J.L. Cherian M.G. Br. J. Cancer. 1995; 71: 712-716Crossref PubMed Scopus (19) Google Scholar). 2K. Ghoshal, unpublished data. The higher basal level of MT-I mRNA in the livers of tumor-bearing rats appears to result from stress caused by the tumor burden. This observation is consistent with the dramatic induction of MT in the livers of normal rats exposed to simple stress (23Ghoshal K. Wang Y. Sheridan J.F. Jacob S.T. J. Biol. Chem. 1998; 273: 27904-27910Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). MT-II mRNA in the hepatoma also remained unaltered after heavy metal treatment (data not shown). BecauseMT-I and MT-II are coordinately induced in all tissues in response to heavy metals, it is not surprising that both these isoforms are not expressed in the hepatoma. Because the hepatoma contains a relatively higher level of GAPDH mRNA, 18 S ribosomal RNA profile in ethidium bromide-stained gel was compared with ascertain equal RNA loading. This experiment was repeated with several different tumor bearing rats, and the results were comparable. As a first step toward the elucidation of the mechanism of noninducibility of MT-I gene in the hepatoma, we measured the DNA binding activity of transacting factors by EMSA. The cis-acting elements and a fewtrans-acting factors that regulate MT gene expression are known. There are several copies (a–f) of metal regulatory element (MRE) that span the upstream promoter of mouse MT-I gene (28Palmiter R.D. Experientia. 1987; 52 (suppl.): 63-80Google Scholar, 29Thiele D.J. Nucleic Acids Res. 1992; 20: 1183-1191Crossref PubMed Scopus (243) Google Scholar). In addition to MREs, the MT-I promoter also contains the binding sites for Sp 1 and MLTF/USF (53Hamer D.H. Annu. Rev. Biochem. 1986; 55: 913-951Crossref PubMed Google Scholar, 54Datta P.K. Jacob S.T. Cell. Mol. Biol. Res. 1993; 39: 439-449PubMed Google Scholar). MTF-1, a 69–86-kDa protein containing six zinc fingers, binds to the MREs in response to heavy metals and activates the genes (45Radtke F. Heuchel R. Georgiev O. Hergersberg M. Gariglio M. Dembic Z. Schaffner W. EMBO J. 1993; 12: 1355-1362Crossref PubMed Scopus (329) Google Scholar, 58Radtke F. Georgiev O. Muller H.P. Brugnera E. Schaffner W. Nucleic Acids Res. 1995; 23: 2277-2286Crossref PubMed Scopus (135) Google Scholar). This transcription factor is necessary for the basal and inducible expression of MT genes by heavy metals and oxygen free radicals (56Dalton T.P. Li Q. Bittel D. Liang L. Andrews G.K. J. Biol. Chem. 1996; 271: 26233-26241Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar,57Cagatay G. Heuchel R. Georgiev O. Lichtlen P. Bluthmann H. Marino S. Aguzzi A. Scaffner W. EMBO J. 1998; 17: 2846-2854Crossref PubMed Scopus (218) Google Scholar). EMSA was used to measure the DNA binding activity of MTF-1 in the liver and the hepatoma nuclear extracts. MRE-d was the oligo of choice in this assay, because MTF-1 exhibited strongest binding affinity for this MRE in vitro (45Radtke F. Heuchel R. Georgiev O. Hergersberg M. Gariglio M. Dembic Z. Schaffner W. EMBO J. 1993; 12: 1355-1362Crossref PubMed Scopus (329) Google Scholar, 55Heuchel R. Radtke F. Georgiev O. Stark G. Aguet M. Schaffner W. EMBO J. 1994; 13: 2805-2870Crossref PubMed Scopus (401) Google Scholar). Because MRE-d has binding sites for both MTF-1 and Sp 1, two DNA-protein complexes were detected in the liver and hepatoma nuclear extracts (Fig. 2 A, lanes 1 and4). To identify the complexes, competitive EMSA was performed with 100-fold molar excess of Sp 1 consensus oligo and MRE-s oligo, a variant of MRE-d in which Sp 1 site is mutated to abolish its binding (45Radtke F. Heuchel R. Georgiev O. Hergersberg M. Gariglio M. Dembic Z. Schaffner W. EMBO J. 1993; 12: 1355-1362Crossref PubMed Scopus (329) Google Scholar). The lower complex in both tissues was competed out with MRE-s oligo (lanes 2 and 5), and the upper complex disappeared with Sp 1 oligo (lanes 3 and6). Because identical amounts of the protein were used in this assay, these data clearly show that both MTF-1 and Sp 1 are more active in the hepatoma than the host liver. The DNA binding activities of MTF-1 and Sp 1 were analyzed with several nuclear extracts prepared from different batches of hepatomas, and the results were reproducible. We also tested whether MTF-1 was activated in the hepatoma in response to zinc. For this purpose we made hepatoma nuclear extract from the animals injected intraperitoneal with ZnSO4 (200 μmol/g) for 4 h and measured the MRE-d binding activity (Fig. 2 B). The complexes formed are specific because their formation could be competed out with an excess of unlabeled MRE-d oligo (lane 3). The slower migrating complex was Sp 1, because its formation was competed by unlabeled Sp 1 consensus oligo (lane 4), and the faster migrating complex was MTF-1, because it could be supershifted with antisera against N-terminal fragment of the protein lackin" @default.
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• W2015120197 title "Suppression of Metallothionein Gene Expression in a Rat Hepatoma Because of Promoter-specific DNA Methylation" @default.
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• W2015120197 doi "https://doi.org/10.1074/jbc.275.1.539" @default.
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