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1.
J Clin Invest ; 122(1): 163-77, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22133874

RESUMEN

DNA methyltransferase 3B (Dnmt3b) belongs to a family of enzymes responsible for methylation of cytosine residues in mammals. DNA methylation contributes to the epigenetic control of gene transcription and is deregulated in virtually all human tumors. To better understand the generation of cancer-specific methylation patterns, we genetically inactivated Dnmt3b in a mouse model of MYC-induced lymphomagenesis. Ablation of Dnmt3b function using a conditional knockout in T cells accelerated lymphomagenesis by increasing cellular proliferation, which suggests that Dnmt3b functions as a tumor suppressor. Global methylation profiling revealed numerous gene promoters as potential targets of Dnmt3b activity, the majority of which were demethylated in Dnmt3b-/- lymphomas, but not in Dnmt3b-/- pretumor thymocytes, implicating Dnmt3b in maintenance of cytosine methylation in cancer. Functional analysis identified the gene Gm128 (which we termed herein methylated in normal thymocytes [Ment]) as a target of Dnmt3b activity. We found that Ment was gradually demethylated and overexpressed during tumor progression in Dnmt3b-/- lymphomas. Similarly, MENT was overexpressed in 67% of human lymphomas, and its transcription inversely correlated with methylation and levels of DNMT3B. Importantly, knockdown of Ment inhibited growth of mouse and human cells, whereas overexpression of Ment provided Dnmt3b+/+ cells with a proliferative advantage. Our findings identify Ment as an enhancer of lymphomagenesis that contributes to the tumor suppressor function of Dnmt3b and suggest it could be a potential target for anticancer therapies.


Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/deficiencia , Linfoma/etiología , Oncogenes , Animales , Proliferación Celular , ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN/genética , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Inestabilidad Genómica , Humanos , Linfoma/genética , Linfoma/metabolismo , Linfoma/patología , Ratones , Ratones Noqueados , Ratones Transgénicos , Linfocitos T/inmunología , Linfocitos T/patología , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética , Regulación hacia Arriba , ADN Metiltransferasa 3B
2.
Int J Biochem Cell Biol ; 43(4): 586-93, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21168523

RESUMEN

Sialyl Lewis X is a tumor-associated antigen frequently found in the advanced cancers. However, the mechanism for the production of this cancer antigen is not entirely clear. The objective of this study is to examine whether epigenetics is involved in the regulation of the formation of this antigen. We observed an increase of sialyl Lewis X in HCT15 cells, a colon cancer cell line, treated with 5-Aza-2'-deoxycytidine. This treatment enhanced the expression of ß-galactoside:α2,3-sialyltransferase 6 gene and sialyl Lewis X on MUC1, and the adherence of these cells to E-selectin under dynamic flow conditions. In addition, 5-Aza-2'-deoxycytidine treatment inhibited methylation of ß-galactoside:α2,3-sialyltransferase 6 gene and siRNA knockdown of this gene drastically reduced sialyl Lewis X without affecting MUC1 expression. We conclude that 5-Aza-2'-deoxycytidine treatment increases sialyl Lewis X on MUC1 by stimulating the ß-galactoside:α2,3-sialyltransferase 6 gene via inhibition of DNA methylation. Increased sialyl Lewis X by 5-Aza-2'-deoxycytidine raises a concern about the safety of this chemotherapeutic drug. In addition, ß-galactoside:α2,3-sialyltransferase 6 gene may be a potential therapeutic target for suppressing tumorigenicity of colon cancer.


Asunto(s)
Azacitidina/análogos & derivados , Inhibidores Enzimáticos/farmacología , Mucina-1/metabolismo , Oligosacáridos/metabolismo , Sialiltransferasas/genética , Azacitidina/farmacología , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Metilación de ADN/efectos de los fármacos , Decitabina , Selectina E/metabolismo , Epigénesis Genética/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucógeno/metabolismo , Glicosilación/efectos de los fármacos , Humanos , Proteínas Inmovilizadas/metabolismo , Peso Molecular , Oligosacáridos/biosíntesis , Oligosacáridos/química , Antígeno Sialil Lewis X , Activación Transcripcional/efectos de los fármacos , beta-Galactosida alfa-2,3-Sialiltransferasa
3.
Biochem Pharmacol ; 78(6): 633-41, 2009 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-19467223

RESUMEN

Aberrant cytosine methylation in promoter regions leads to gene silencing associated with cancer progression. A number of DNA methyltransferase inhibitors are known to reactivate silenced genes; including 5-azacytidine and 2-(1H)-pyrimidinone riboside (zebularine). Zebularine is a more stable, less cytotoxic inhibitor compared to 5-azacytidine. To determine the mechanistic basis for this difference, we carried out a detailed comparisons of the interaction between purified DNA methyltransferases and oligodeoxyribonucleotides (ODNs) containing either 5-azacytosine or 2-(1H)-pyrimidinone in place of the cytosine targeted for methylation. When incorporated into small ODNs, the rate of C5 DNA methyltransferase inhibition by both nucleosides is essentially identical. However, the stability and reversibility of the enzyme complex in the absence and presence of cofactor differs. 5-Azacytosine ODNs form complexes with C5 DNA methyltransferases that are irreversible when the 5-azacytosine ring is intact. ODNs containing 2-(1H)-pyrimidinone at the enzymatic target site are competitive inhibitors of both prokaryotic and mammalian DNA C5 methyltransferases. We determined that the ternary complexes between the enzymes, 2-(1H)-pyrimidinone inhibitor, and the cofactor S-adenosyl methionine are maintained through the formation of a reversible covalent interaction. The differing stability and reversibility of the covalent bonds may partially account for the observed differences in cytotoxicity between zebularine and 5-azacytidine inhibitors.


Asunto(s)
Citidina/análogos & derivados , Citosina/análogos & derivados , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , ADN/farmacología , Oligodesoxirribonucleótidos/farmacología , Sitios de Unión , Citidina/farmacología , Citosina/farmacología , ADN/química , ADN/efectos de los fármacos , ADN/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Conformación Proteica , Relación Estructura-Actividad
4.
J Nutr ; 138(12): 2316-22, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19022951

RESUMEN

Transposable elements such as long terminal repeats (LTR) constitute approximately 45% of the human genome; transposition events impair genome stability. Fifty-four promoter-active retrotransposons have been identified in humans. Epigenetic mechanisms are important for transcriptional repression of retrotransposons, preventing transposition events, and abnormal regulation of genes. Here, we demonstrate that the covalent binding of the vitamin biotin to lysine-12 in histone H4 (H4K12bio) and lysine-9 in histone H2A (H2AK9bio), mediated by holocarboxylase synthetase (HCS), is an epigenetic mechanism to repress retrotransposon transcription in human and mouse cell lines and in primary cells from a human supplementation study. Abundance of H4K12bio and H2AK9bio at intact retrotransposons and a solitary LTR depended on biotin supply and HCS activity and was inversely linked with the abundance of LTR transcripts. Knockdown of HCS in Drosophila melanogaster enhances retrotransposition in the germline. Importantly, we demonstrated that depletion of H4K12bio and H2AK9bio in biotin-deficient cells correlates with increased production of viral particles and transposition events and ultimately decreases chromosomal stability. Collectively, this study reveals a novel diet-dependent epigenetic mechanism that could affect cancer risk.


Asunto(s)
Elementos Transponibles de ADN , Histonas/química , Histonas/metabolismo , Adulto , Animales , Biotina/administración & dosificación , Biotinilación , Ligasas de Carbono-Nitrógeno/antagonistas & inhibidores , Ligasas de Carbono-Nitrógeno/genética , Ligasas de Carbono-Nitrógeno/metabolismo , Línea Celular , Aberraciones Cromosómicas , Citosina/metabolismo , Metilación de ADN , Suplementos Dietéticos , Drosophila melanogaster , Epigénesis Genética , Femenino , Humanos , Células Jurkat , Masculino , Virus del Tumor Mamario del Ratón/efectos de los fármacos , Virus del Tumor Mamario del Ratón/fisiología , Ratones , Persona de Mediana Edad , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Secuencias Repetidas Terminales , Transcripción Genética/efectos de los fármacos , Ensamble de Virus/efectos de los fármacos , Adulto Joven
5.
J Mol Biol ; 379(3): 492-504, 2008 Jun 06.
Artículo en Inglés | MEDLINE | ID: mdl-18452947

RESUMEN

Methylation of cytosine residues in CpG dinucleotides plays an important role in epigenetic regulation of gene expression and chromatin structure/stability in higher eukaryotes. DNA methylation patterns are established and maintained at CpG dinucleotides by DNA methyltransferases (Dnmt1, Dnmt3a, and Dnmt3b). In mammals and many other eukaryotes, the CpG dinucleotide is underrepresented in the genome. This loss is postulated to be the result of unrepaired deamination of cytosine and 5-methylcytosine to uracil and thymine, respectively. Two thymine glycosylases are believed to reduce the impact of 5-methylcytosine deamination. G/T mismatch-specific thymine-DNA glycosylase (Tdg) and methyl-CpG binding domain protein 4 can both excise uracil or thymine at U.G and T.G mismatches to initiate base excision repair. Here, we report the characterization of interactions between Dnmt3b and both Tdg and methyl-CpG binding domain protein 4. Our results demonstrate (1) that both Tdg and Dnmt3b are colocalized to heterochromatin and (2) reduction of T.G mismatch repair efficiency upon loss of DNA methyltransferase expression, as well as a requirement for an RNA component for correct T.G mismatch repair.


Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilasas de Modificación del ADN/metabolismo , Reparación del ADN , Endodesoxirribonucleasas/metabolismo , ARN/metabolismo , Timina ADN Glicosilasa/metabolismo , Animales , Secuencia de Bases , Línea Celular , ADN (Citosina-5-)-Metiltransferasas/química , ADN (Citosina-5-)-Metiltransferasas/genética , Daño del ADN , Metilasas de Modificación del ADN/genética , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/genética , Humanos , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos/genética , Oligodesoxirribonucleótidos/metabolismo , ARN/genética , Timina ADN Glicosilasa/química , Timina ADN Glicosilasa/genética , Técnicas del Sistema de Dos Híbridos , ADN Metiltransferasa 3B
6.
Zhonghua Bing Li Xue Za Zhi ; 36(4): 253-8, 2007 Apr.
Artículo en Chino | MEDLINE | ID: mdl-17706117

RESUMEN

OBJECTIVE: To investigate the role of WT1 gene in breast carcinogenesis by analyses of the promoter methylation status and mRNA expression of WT1 gene in MCF10 model system of breast cancer progression. METHODS: Methylation specific PCR and sodium bisufite genomic sequencing were employed to detect methylation status of WT1 promoter in normal breast tissue, traditional breast cancer cell line MCF7 and MCF10 model series, including MCF10A (breast hyperplastic cell line, non-tumorigenic), MCF10AT (pre-malignant cell line, forming slowly progressing hyper and dysplastic lesions), MCF10DCIS.com (breast ductal carcinoma in situ cell line, forming ductal carcinoma in situ), and three invasive cell lines with metastatic potential (MCF10CA1a, MCF10CA1d, and MCF10CA1h). Real time reverse transcription PCR assay was used to determine the mRNA expression levels of WT1 in various cell lines. RESULTS: Hypermethylation of WT1 promoter was identified in MCF7 and all MCF10 model cell lines (MCF10A, MCF10AT, MCF10DCIS.com, MCF10CA1a, MCF10CA1d, and MCF10CA1h). Unexpectedly, an increased expression of WT1 mRNA was found in all MCF10 cell lines and MCF7 comparing with normal breast tissue [folds of overexpression: 3.23 (MCF10A), 1.94 (MCF10AT), 4.20 (MCF10CA1a), 1.53 (MCF10CA1d), 4.20 (MCF10CA1h), 4.35 (MCF10DCIS) and 28.69 (MCF7)]. CONCLUSIONS: Promoter methylation does not silence the mRNA expression of WT1 during the development of breast cancer. Overexpression of WT1 occurs in the early stages of breast cancer development, suggesting its role as an oncogene rather than a tumor suppressor gene.


Asunto(s)
Neoplasias de la Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Metilación de ADN , Proteínas WT1/metabolismo , Secuencia de Bases , Mama/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/patología , Carcinoma Intraductal no Infiltrante/genética , Carcinoma Intraductal no Infiltrante/metabolismo , Carcinoma Intraductal no Infiltrante/patología , Línea Celular Tumoral , ADN de Neoplasias/genética , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Hiperplasia/genética , Hiperplasia/metabolismo , Hiperplasia/patología , Datos de Secuencia Molecular , Lesiones Precancerosas/genética , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patología , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Proteínas WT1/genética
7.
Zhonghua Bing Li Xue Za Zhi ; 35(1): 32-6, 2006 Jan.
Artículo en Chino | MEDLINE | ID: mdl-16608647

RESUMEN

OBJECTIVE: To investigate the promoter methylation status and mRNA expression of APC gene in MCF10 model of breast cancer progression. METHODS: Methylation specific PCR and sodium bisufite genomic sequencing were employed to detect the methylation status of APC promoter 1A in normal breast tissues, conventional breast cancer cell line MCF-7 and MCF10 model cell lines including MCF10A (breast hyperplastic cell line, non-tumorigenic), MCF10AT (pre-malignant cell lines, producing slowly progressing hyperplastic and dysplastic lesions), MCF10DCIS.com (breast ductal carcinoma in-situ cell line, producing ductal carcinoma in-situ), MCF10CA1a, MCF10CA1d, MCF10CA1h cell lines (invasive breast carcinoma cell line, forming aggressive tumors of different morphology and metastatic potential). In addition, mRNA expression of APC was determined by reverse transcriptase PCR and real-time PCR assays. RESULTS: Hypomethylation of APC promoter 1A was identified in hyperplastic cell line MCF10A, pre-malignant cell line MCF10AT, ductal carcinoma in-situ cell line MCF10DCIS.com, invasive carcinoma cell lines MCF10CA1a, MCF10CA1d, MCF10CA1h and normal breast tissue. MCF-7 showed partial methylation at the promoter. Statistically significant reduction of APC mRNA expression was not found in all MCF10 cell lines and MCF-7, compared with that of normal breast tissue (MCF10AT, MCF10CA1a, MCF10CA1d, MCF10CA1h and MCF10DCIS.com showed reduced mRNA expressions of APC at 0.27, 0.96, 1.78, 2.70, and 2.03 times respectively. MCF10A and MCF-7 even showed an increase of APC mRNA expression at 0.02 and 0.33 times, respectively). CONCLUSION: The aberrant promoter methylation of APC is not related to the breast cancer progression, at least in the MCF10 model system.


Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon/genética , Neoplasias de la Mama/metabolismo , Metilación de ADN , Lesiones Precancerosas/metabolismo , Regiones Promotoras Genéticas/genética , Proteína de la Poliposis Adenomatosa del Colon/biosíntesis , Mama/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Genes APC , Humanos , Hiperplasia/genética , Hiperplasia/metabolismo , Hiperplasia/patología , Lesiones Precancerosas/genética , Lesiones Precancerosas/patología , ARN Mensajero/biosíntesis , ARN Mensajero/genética
8.
Clin Cancer Res ; 11(19 Pt 1): 7033-41, 2005 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-16203797

RESUMEN

PURPOSE: We have previously shown the reactivation of some methylation-silenced genes in cancer cells by (-)-epigallocatechin-3-gallate, the major polyphenol from green tea. To determine whether other polyphenolic compounds have similar activities, we studied the effects of soy isoflavones on DNA methylation. EXPERIMENTAL DESIGN: Enzyme assay was used to determine the inhibitory effect of genistein on DNA methyltransferase activity in nuclear extracts and purified recombinant enzyme. Methylation-specific PCR and quantitative real-time PCR were employed to examine the DNA methylation and gene expression status of retinoic acid receptor beta (RARbeta), p16INK4a, and O6-methylguanine methyltransferase (MGMT) in KYSE 510 esophageal squamous cell carcinoma cells treated with genistein alone or in combination with trichostatin, sulforaphane, or 2'-deoxy-5-aza-cytidine (5-aza-dCyd). RESULTS: Genistein (2-20 micromol/L) reversed DNA hypermethylation and reactivated RARbeta, p16INK4a, and MGMT in KYSE 510 cells. Genistein also inhibited cell growth at these concentrations. Reversal of DNA hypermethylation and reactivation of RARbeta by genistein were also observed in KYSE 150 cells and prostate cancer LNCaP and PC3 cells. Genistein (20-50 micromol/L) dose-dependently inhibited DNA methyltransferase activity, showing substrate- and methyl donor-dependent inhibition. Biochanin A and daidzein were less effective in inhibiting DNA methyltransferase activity, in reactivating RARbeta, and in inhibiting cancer cell growth. In combination with trichostatin, sulforaphane, or 5-aza-dCyd, genistein enhanced reactivation of these genes and inhibition of cell growth. CONCLUSIONS: These results indicate that genistein and related soy isoflavones reactivate methylation-silenced genes, partially through a direct inhibition of DNA methyltransferase, which may contribute to the chemopreventive activity of dietary isoflavones.


Asunto(s)
Anticarcinógenos/farmacología , Antineoplásicos/farmacología , Carcinoma de Células Escamosas/tratamiento farmacológico , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Metilación de ADN , Neoplasias Esofágicas/tratamiento farmacológico , Genisteína/farmacología , Glycine max/metabolismo , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Receptores de Ácido Retinoico/metabolismo , Azacitidina/análogos & derivados , Azacitidina/farmacología , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Proliferación Celular , Decitabina , Relación Dosis-Respuesta a Droga , Neoplasias Esofágicas/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Isoflavonas/química , Isoflavonas/farmacología , Isotiocianatos , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sulfitos/farmacología , Sulfóxidos , , Tiocianatos/farmacología
10.
Int J Cancer ; 108(5): 672-8, 2004 Feb 20.
Artículo en Inglés | MEDLINE | ID: mdl-14696093

RESUMEN

Members of Shc family conventionally serve as critical adaptors in tyrosine phosphorylation signal transduction pathways. p66(Shc) protein, a member of Shc family, is predominantly expressed in epithelial cells, whereas the regulation of its expression remains an enigma. We describe the effect of steroid hormones on the protein level of p66(Shc) and growth stimulation in hormone-sensitive human prostate, testicular and breast cancer cells. In DHT-treated androgen-sensitive prostate cancer LNCaP C-33 cells, the protein level of p66(Shc) was elevated by approximately 3-fold, correlating with increased cell growth. This DHT effect on p66(Shc) protein level and growth regulation was also observed in another androgen-sensitive prostate cancer cell line MDA PCa2b as well as 2 testicular cancer cell lines, Tera-1 and Tera-2 cells. Similarly, the female sex hormone estrogen had a stimulating effect on p66(Shc) protein level and proliferation in estrogen-sensitive MCF-7 breast cancer cells. The upregulation of p66(Shc) protein level by DHT was competitively abolished by Casodex, an androgen antagonist used to treat prostate cancer. Moreover, immunohistochemical analyses showed that the p66(Shc) protein level was significantly higher in primary prostate tumors than in adjacent non-cancerous cells (p < 0.05). The data collectively indicate that p66(Shc) protein levels correlate with steroid hormone-stimulated cell growth and prostate carcinogenesis.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular/biosíntesis , Dihidrotestosterona/farmacología , Neoplasias Hormono-Dependientes/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Mama/metabolismo , División Celular , Estrógenos/farmacología , Humanos , Masculino , Fosforilación , Próstata/metabolismo , Proteínas Adaptadoras de la Señalización Shc , Transducción de Señal , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Neoplasias Testiculares/metabolismo , Células Tumorales Cultivadas , Regulación hacia Arriba
11.
Ann N Y Acad Sci ; 1002: 154-64, 2003 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-14751833

RESUMEN

A short oligodeoxynucleotide (ODN) with 2-(1H)-pyrimidinone at the HhaI DNA methyltransferase target site (GCGC) is shown to induce a level of inhibition of methyl transfer and thermal stability of the complex with the enzyme identical to that achieved with a similar ODN substituted with 5-azacytosine. The drugs responsible for these effects-zebularine and 5-azacytidine/2'-deoxy-5-azacytidine-are contrasted in terms of chemical stability and possible metabolic activation by a brief structure-activity analysis.


Asunto(s)
Antineoplásicos/metabolismo , ADN-Citosina Metilasas/antagonistas & inhibidores , Nucleósidos de Pirimidina/metabolismo , Citidina/análogos & derivados , Citidina Desaminasa/antagonistas & inhibidores , ADN/metabolismo , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , Estructura Terciaria de Proteína , Relación Estructura-Actividad
12.
J Mol Biol ; 323(1): 53-67, 2002 Oct 11.
Artículo en Inglés | MEDLINE | ID: mdl-12368098

RESUMEN

The presence of 5-azacytosine (ZCyt) residues in DNA leads to potent inhibition of DNA (cytosine-C5) methyltranferases (C5-MTases) in vivo and in vitro. Enzymatic methylation of cytosine in mammalian DNA is an epigenetic modification that can alter gene activity and chromosomal stability, influencing both differentiation and tumorigenesis. Thus, it is important to understand the critical mechanistic determinants of ZCyt's inhibitory action. Although several DNA C5-MTases have been reported to undergo essentially irreversible binding to ZCyt in DNA, there is little agreement as to the role of AdoMet and/or methyl transfer in stabilizing enzyme interactions with ZCyt. Our results demonstrate that formation of stable complexes between HhaI methyltransferase (M.HhaI) and oligodeoxyribonucleotides containing ZCyt at the target position for methylation (ZCyt-ODNs) occurs in both the absence and presence of co-factors, AdoMet and AdoHcy. Both binary and ternary complexes survive SDS-PAGE under reducing conditions and take on a compact conformation that increases their electrophoretic mobility in comparison to free M.HhaI. Since methyl transfer can occur only in the presence of AdoMet, these results suggest (1) that the inhibitory capacity of ZCyt in DNA is based on its ability to induce a stable, tightly closed conformation of M.HhaI that prevents DNA and co-factor release and (2) that methylation of ZCyt in DNA is not required for inhibition of M.HhaI.


Asunto(s)
Azacitidina/análogos & derivados , Azacitidina/química , ADN-Citosina Metilasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Oligodesoxirribonucleótidos/farmacología , Secuencia de Bases , Metilación de ADN , ADN-Citosina Metilasas/química , Decitabina , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/química , Oligodesoxirribonucleótidos/química , Conformación Proteica
13.
Oncogene ; 21(35): 5483-95, 2002 Aug 12.
Artículo en Inglés | MEDLINE | ID: mdl-12154409

RESUMEN

5-Azacytidine was first synthesized almost 40 years ago. It was demonstrated to have a wide range of anti-metabolic activities when tested against cultured cancer cells and to be an effective chemotherapeutic agent for acute myelogenous leukemia. However, because of 5-azacytidine's general toxicity, other nucleoside analogs were favored as therapeutics. The finding that 5-azacytidine was incorporated into DNA and that, when present in DNA, it inhibited DNA methylation, led to widespread use of 5-azacytidine and 5-aza-2'-deoxycytidine (Decitabine) to demonstrate the correlation between loss of methylation in specific gene regions and activation of the associated genes. There is now a revived interest in the use of Decitabine as a therapeutic agent for cancers in which epigenetic silencing of critical regulatory genes has occurred. Here, the current status of our understanding of the mechanism(s) by which 5-azacytosine residues in DNA inhibit DNA methylation is reviewed with an emphasis on the interactions of these residues with bacterial and mammalian DNA (cytosine-C5) methyltransferases. The implications of these mechanistic studies for development of less toxic inhibitors of DNA methylation are discussed.


Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Azacitidina/análogos & derivados , Azacitidina/farmacología , Metilación de ADN/efectos de los fármacos , ADN de Neoplasias/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Neoplasias/terapia , Metilasas de Modificación del ADN/antagonistas & inhibidores , Decitabina , Humanos , Neoplasias/enzimología
14.
Protein Expr Purif ; 25(1): 31-40, 2002 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-12071696

RESUMEN

Enzymatic DNA methylation of carbon 5 of cytosines is an epigenetic modification that plays a role in regulating gene expression, differentiation, and tumorigenesis. DNA (cytosine-C5)-methyltransferase-1 is the enzyme responsible for maintaining established methylation patterns during replication in mammalian cells. It is composed of a large ( approximately 1100 amino acids (a.a.)) amino-terminal region containing many putative regulatory domains and a smaller ( approximately 500 a.a.) carboxy-terminal region containing conserved, catalytic domains. In this study, murine DNA (cytosine C5)-methyltransferase-1, fused to an amino-terminal hexahistidine tag, was expressed by infecting Spodoptera frugiperda cells for 46 h with a recombinant baculovirus carrying the DNA (cytosine-C5)-methyltransferase-1 cDNA. A total of 3 x 10(8) infected S. frugiperda cells yielded approximately 1 mg of full-length, hexahistidine-tagged DNA (cytosine-C5)-methyltransferase-1, which was purified approximately 450-fold from RNase-treated S. frugiperda cell extracts by nickel affinity chromatography. The characterization of hexahistidine-tagged DNA (cytosine-C5)-methyltransferase-1 through DNA methylation and inhibitor-binding assays indicated that the purified enzyme had at least a 30-fold higher catalytic efficiency with hemimethylated double-stranded oligodeoxyribonucleotide substrates than unmethylated substrates and was most active with small oligodeoxyribonucleotide substrates with a capacity for forming stem-loop structures. The expression and purification procedures reported here differ significantly from the original reports of baculovirus-mediated hexahistidine-tagged DNA (cytosine-C5)-methyltransferase-1 expression and purification by nickel affinity chromatography and provide a consistent yield of active enzyme.


Asunto(s)
Baculoviridae/metabolismo , ADN (Citosina-5-)-Metiltransferasas/biosíntesis , ADN (Citosina-5-)-Metiltransferasas/aislamiento & purificación , Histidina/metabolismo , Animales , Sitios de Unión , Western Blotting , Línea Celular , Cromatografía de Afinidad , Metilación de ADN , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Flucitosina/metabolismo , Insectos , Cinética , Ratones , Oligonucleótidos/metabolismo , Spodoptera , Factores de Tiempo
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