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PubMed2005

A novel methyltransferase required for the formation of the hypermodified nucleoside wybutosine in eucaryotic tRNA.

We demonstrate that the product of the yeast open reading frame YML005w is required for wybutosine (yW) formation in the phenylalanine-accepting tRNA of the yeast Saccharomyces cerevisiae. tRNA isolated from a deletion mutant of the YML005w gene accumulates 4-demethylwyosine (ImG-14), a precursor lacking three of the methyl groups of the yW hypermodified base. Since the amino acid sequence of the YML005w gene contains the signature motifs of the seven beta-strand methyltransferases, we now designate the gene TRM12 for tRNA methyltransferase. Using pulse-chase labeling of intact yeast cells with S-adenosyl-L-[methyl-(3)H]methionine, we show that the methylesterified form of yW is metabolically stable.

Amino Acid Sequence↗
PubMed2006

5-Aza-2'-deoxycytidine and depsipeptide synergistically induce expression of BIK (BCL2-interacting killer).

DNA methylation and histone acetylation are main epigenetic events regulating gene expression, serving as anticancer drug targets. A combination of the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine with the histone deacetylase inhibitor depsipeptide synergistically induces apoptosis. To characterize genes involved in this process, we measured expression of 376 apoptosis-related genes with microarrays after treatment with the two inhibitors alone or in combination. The pro-apoptotic BIK (Bcl2-interacting killer) was the only gene synergistically upregulated in all four cancer cell lines tested (A549, PC-3, TK-10, and UO-31). BIK induction was confirmed by RT-PCR and Western blots. Histone acetylation of the BIK promoter region increased with depsipeptide treatment but was not further affected by 5-aza-2'-deoxycytidine. In summary, synergistic upregulation of pro-apoptotic BIK-previously shown to suppress tumor growth-appears to play a critical role in anticancer effects of 5-aza-2'-deoxycytidine plus depsipeptide.

Acetylation↗
PubMed2004

DNA methyltransferase inhibitors in myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) resulting from a clonal stem cell is a heterogeneous disease that complicates therapeutic decisions. Most patients are of advanced age with attendant comorbities, making treatment choices difficult. Current treatment options include bone marrow transplant, which appears to be the only curative option, and supportive care. In elderly patients, risks for transplant are high and supportive-care treatments are ineffective. However, considerable progress has been made in understanding the pathology of MDS, which results from a series of progressive chromosomal assaults that lead to a release of various cytokines, loss of tumor suppressor genes, and changes in signal transduction pathways and in immune pathways. The neoplastic clone does not appear to mature; it stays in a fixed state of differentiation beyond which these cells do not progress. Hypermethylation of specific DNA sequences, which results in silencing transcription of proteins involved, has been implicated in this lack of differentiation. New clinical discoveries include the potential to block methylation, release transcriptional inactivation, and stimulate the normal myeloid cells to resume growth and differentiation. Therapies aimed at these new findings hold promise for safer treatment protocols and improved outcomes. One of these agents, azacitidine, is a nucleoside analog that has shown promising efficacy in high-risk MDS patients. Phase III studies have shown significant improvement in survival and quality of life with minimal side effects. Prolonged administration of azacitidine results in reactivation of normal hematopoiesis by its effect on inhibiting DNA methylation.

Azacitidine↗
PubMed2005

5-Aza-2'-deoxycytidine induces retinoic acid receptor-beta(2) demethylation and growth inhibition in esophageal squamous carcinoma cells.

Esophageal squamous cell carcinoma (ESCC) is one of the most common tumors in human. Previous studies showed that multiple genetic and epigenetic alterations involved in carcinogenesis of esophagus, whereas the molecular mechanisms are poorly understood. So far, more and more ESCC-related genes have been found and retinoic acid receptor beta(2) (RARbeta(2)) is such a gene which was recognized as a putative tumor suppressor gene since reduced RARbeta(2) mRNA expression has been observed in several solid tumors, including ESCC. A growing evidence indicated that RARbeta(2) was required for the growth inhibitory effect of retinoic acid (RA). However, the molecular mechanism of its inactivation remained obscure in ESCC. The RARbeta2 methylation status was assessed by methylation-specific PCR (MSP) in 12 ESCC cell lines and compared with their mRNA and protein expression level. Bisulfite sequencing of RARbeta(2) promoter region was performed to confirm the MSP results. After 5-aza-2'-deoxycytidine (5-aza-dc) treatment the expression of RARbeta(2) was reversed in two RARbeta(2)-downregulated cell lines. Therefore, hypermethylation of the promoter regions of RARbeta2 gene is a major mechanism of transcriptional inactivation and might be involved in tumor development of esophagus in some ESCC cell lines suggesting that multiple mechanisms contribute to the loss of RARbeta(2) expression in ESCC cell lines. Furthermore, the methylation status of RARbeta(2) promoter region and its expression was analyzed in 51 ESCC tissue samples with their adjacent normal epithelia and two normal esophageal epithelia. The results showed that there was a statistically significant correlation between methylation status of RARbeta(2) and tumor grade; Moreover, a relationship between methylation status and decreased RARbeta(2) expression was found only in G(2) stage tumors. After 5-aza-dc treatment, RARbeta(2) restoration was accompanied by growth inhibition and this might be one of the mechanisms but not the only mechanism for the tumor cell growth inhibition by 5-aza-dc. This study may have clinical applications for ESCC therapy and prevention.

Adult↗
PubMed2006

Lytic induction and apoptosis of Epstein-Barr virus-associated gastric cancer cell line with epigenetic modifiers and ganciclovir.

Epstein-Barr virus (EBV) is an oncogenic herpes virus. EBV gene transcription is regulated by an epigenetic mechanism to establish a persistent infection and to evade the host immune system. We found that low concentrations of epigenetic modifying agents, 5-aza-2'-deoxycytidine (5-aza-CdR) or trichostatin A (TSA), induced the expression of BMRF1, BZLF1, and BRLF1 genes, which are found in the lytic form of the virus, in an EBV-positive gastric cancer cell line. This effect did not involve PI3 kinase, MAP/ERK kinase, protein kinase C delta, or p38 MAPK signaling pathway. The cytotoxic effect of ganciclovir (GCV) was enhanced after the lytic induction by epigenetic modifiers, and the combination of GCV and epigenetic modifiers induced apoptosis, which is dependent on caspases. In conclusion, the combination of GCV with 5-aza-CdR or TSA might be a useful therapeutic strategy for EBV-induced human gastric cancer.

Apoptosis↗
PubMed2005

Controlling DNA methylation: many roads to one modification.

Genetic, biochemical and cytological studies on DNA methylation in several eukaryotic organisms have resulted in leaps of understanding in the past three years. Discoveries of mechanistic links between DNA methylation and histone methylation, and between these processes and RNA interference (RNAi) machineries have reinvigorated the field. The details of the connections between DNA methylation, histone modifications and RNA silencing remain to be elucidated, but it is already clear that no single pathway accounts for all DNA methylation found in eukaryotes. Rather, different taxa use one or more of several general mechanisms to control methylation. Despite recent progress, classic questions remain, including: What are the signals for DNA methylation? Are "de novo" and "maintenance" methylation truly separate processes? How is DNA methylation regulated?

Chromatin↗
PubMed2005

Quantitative determination of zebularine (NSC 309132), a DNA methyltransferase inhibitor, and three metabolites in murine plasma by high-performance liquid chromatography coupled with on-line radioactivity detection.

The metabolism of zebularine (NSC 309132), a novel agent that inhibits DNA methyltransferases, is still uncharacterized. To examine the in vivo metabolism of zebularine, an analytical method was developed and validated (based on FDA guidelines) to quantitate 2-[(14)C]-zebularine and its major metabolites in murine plasma. Zebularine and its metabolites uridine, uracil and dihydrouracil were baseline-separated based on hydrophilic interaction chromatography by using an amino column. The assay was accurate and precise in the concentration ranges of 5.0-100 microg/mL for zebularine, 2.5-50 microg/mL for uridine, 1.0-10 microg/mL for uracil and 0.5-5.0 microg/mL for dihydrouracil. This assay is being used to quantitate zebularine and its metabolites in ongoing pharmacokinetic studies of zebularine.

Animals↗
PubMed2006

Cells lytically infected with Epstein-Barr virus are detected and separable by immunoglobulins from EBV-seropositive individuals.

The role of Epstein-Barr virus (EBV) lytic cycle gene expression in lymphocytes in the pathogenesis of EBV-associated diseases is incompletely understood. The ability to physically separate lytically induced from latently infected cells from the same population and to examine them in parallel would significantly enhance understanding of the viral, cellular, and environmental factors that govern susceptibility of an EBV-infected cell to lytic cycle induction stimuli at the single cell level. This study demonstrates, using a flow cytometry-based system, that human serum immunoglobulins from individuals immune to EBV reproducibly discriminate between and can be used to physically separate lytically induced from latently infected B cells. Using this new quantitative and sensitive technique, two novel observations about lytic cycle activation were made. First, the kinetics of lytic cycle activation by histone deacetylase inhibitors is more rapid than induction by a DNA methyl transferase inhibitor. Second, butyrate-treated cells which are initially refractory to lytic cycle activation can be induced upon subsequent exposure to the inducing agent. Therefore, susceptibility to lytic cycle induction of a latently infected B cell is governed by environmental and physiologic factors and not by permanent cellular or viral genetic changes.

Adolescent↗
PubMed2005

Up-regulation of DNA methyltransferases DNMT1, 3A, and 3B in myelodysplastic syndrome.

Analyzing bone marrow trephines from (myelodysplastic syndrome) MDS patients we show for the first time strong over-expression of the DNA methyltransferases 1, and 3A (and 3B to a much lesser extent) in the myelodysplastic syndrome FAB subtypes refractory anaemia (RA) and refractory anaemia with excess of blasts (RAEB). The increase in mRNA expression was much less pronounced in refractory anaemia with ringed sideroblasts (RARS). Classification according to the new WHO guidelines revealed distinct differences between RCMD and RARS. This elevated mRNA expression most probably contributes to the frequently found aberrant hypermethylation in MDS and might explain the promising clinical response of MDS patients treated with DNMT inhibitors.

Biopsy, Needle↗
PubMed2005

Multi-gene epigenetic silencing of tumor suppressor genes in T-cell lymphoma cells; delayed expression of the p16 protein upon reversal of the silencing.

To understand better T-cell lymphomagenesis, we examined promoter CpG methylation and mRNA expression of closely related genes encoding p16, p15, and p14 tumor suppressor genes in cultured malignant T-cells that were derived from cutaneous, adult type, and anaplastic lymphoma kinase (ALK)-expressing T-cell lymphomas. p16 gene was epigenetically silenced in all but one of the 10 malignant T-cell lines examined, p15 gene silenced in roughly half of the lines, and p14 was the least frequently affected. Extensive methylation of the p16 promoter was seen in six out of 10 cutaneous T-cell lymphoma patient samples and corresponded with lack of p16 protein expression in the cases examined. Treatment of cultured T-cells with the DNA methyltransferase inhibitor, 5-aza-2-deoxy-cytidine, resulted in reversal of the p16 gene silencing. However, expression of p16 protein was delayed in relationship to p16 promoter demethylation and required up to 3 weeks to occur, seemingly reflecting late activation of the p16 gene. These findings indicate that epigenetic silencing affects in T-cell malignancies, often simultaneously, several tumor suppressor genes that impact on key cell functions. The observed differential silencing of p16 and p14, and to a lesser degree p15 gene, indicates that the silencing is governed by precise, promoter region-specific mechanisms. The study provides also further rationale for treatment of at least some types of T-cell lymphomas with DNA methyltransferase inhibitors to target the epigenetically silenced tumor suppressor genes.

Adult↗
PubMed2006

Reversal of p15/INK4b hypermethylation in AML1/ETO-positive and -negative myeloid leukemia cell lines.

In vitro and in vivo, myeloid leukemic and preleukemic cells exhibit variable sensitivity to the antiproliferative and proapoptotic effects induced already at low concentrations of DNA methyltransferase (DNMT) inhibitors. The molecular mechanisms underlying this variable sensitivity of leukemic blasts to azanucleosides such as 5-azacytidine and 5-aza-2'-deoxycytidine (DAC) may involve modifier effects of specific fusion proteins such as AML1/ETO. The cyclin-dependent kinase inhibitor p15/INK4b is one potential target of DNA demethylating activity in AML and MDS where it is frequently silenced by hypermethylation. To study sensitivity to DAC in myeloid leukemia cells, we chose the myeloid cell lines Kasumi-1 (expressing AML1/ETO), KG-1 and KG-1a (both AML1/ETO-negative) all of which a highly methylated p15/INK4b gene. Treatment with DAC resulted in dose-dependent regional demethylation of p15/INK4b in Kasumi-1 and KG-1, but only to a modest degree in KG-1a cells. Demethylation was associated with induction of p15/INK4b protein expression. Growth-inhibitory and proapoptotic activity of DAC was significantly higher in Kasumi-1 than in KG-1a cells, and sensitization of cells to a cooperating effect of All-trans retinoic acid and of the histone deacetylase (HDAC) inhibitor Trichostatin A was observed. DAC-induced growth inhibition and apoptosis were enhanced when AML1/ETO was conditionally expressed in AML1/ETO-negative U-937 cells. In conclusion, hypomethylation and reactivation of p15/INK4b in myeloid cell lines are among the molecular events associated with DAC-induced growth arrest and apoptosis. Further studies of AML1/ETO as a modifier of the epigenotype and sensitivity of myeloid cells to inhibitors of DNMTs and HDACs appear warranted.

Azacitidine↗
PubMed2006

Alteration of DNA methyltransferases contributes to 5'CpG methylation and poor prognosis in lung cancer.

Overexpression of DNA methyltransferases DNMT1, DNMT3a and DNMT3b has been reported in various cancers. However, physical binding of DNA methyltransferase (DNMT) to the hypermethylated promoter of tumor suppressor genes (TSGs) has never been demonstrated in tumor tissues. In addition, alteration of DNMT at the protein level has never been reported in the same series of cancer patients. By immunohistochemical analysis, we demonstrated that DNMT1, DNMT3a and DNMT3b proteins were highly expressed in a coordinate manner in lung tumors, particularly in smokers (P=0.037, by the Fisher exact test). Patients with DNMT1 overexpression had a trend of poorer prognosis than those without such overexpression, and this prognostic significance was apparent in squamous carcinoma (SQ) patients (P=0.041, by the log-rank test). Both DNMT1 and DNMT3b overexpressions correlated with hypermethylation in the TSG promoters, especially among smoking SQ patients (P=0.012). To further explore the molecular mechanisms between altered TSGs promoter methylation and overexpression of DNMTs protein, we performed a tissue chromatin-immunoprecipitation polymerase chain reaction assay for lung tumors and showed that the methylated FHIT, p16(INK4a) and RARbeta promoters were bound by both DNMT protein and methyl-CpG-binding protein 2. These data suggest that overexpression and strong binding of various DNMTs may result in promoter hypermethylation of multiple TSGs and ultimately lead to lung tumorigenesis and poor prognosis.

Blotting, Western↗
PubMed2005

Chromatin modifiers that control plant development.

The different cell types of a multicellular organism express different sets of genes. Although this is one of the oldest paradigms of developmental genetics, how different patterns of gene expression are established and maintained during subsequent cell division is an active topic of research. Chromatin modifiers play an essential role in controlling gene expression and in establishing epigenetic marks that can be inherited. During the past few years, large number of putative chromatin-associated proteins have been uncovered as controllers of meristem organization and activity, phase transition, and gametophyte and embryo development.

Chromatin↗
PubMed2004

Microarray analysis of gene regulation in the Hepa1c1c7 cell line following exposure to the DNA methylation inhibitor 5-aza-2'-deoxycytidine and 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Differential expression of various genes was observed in the Hepa1c1c7 cell line following exposure to the DNA methylation inhibitor 5-aza-2'-deoxycytidine (AzaC) and to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). AzaC treatment generally affected genes induced by TCDD by modulating their induction levels. Induction of several genes, such as receptor (calcitonin) activity modifying protein 3 (Ramp3) by TCDD was enhanced by AzaC, although AzaC by itself was without effect. Some genes, such as frequently rearranged in advanced T-cell lymphomas (Frat1), were up-regulated by AzaC alone, with this induction being negatively affected by TCDD. Other genes were induced by AzaC, TCDD and their co-treatment. In contrast, many genes such as small proline-rich protein 1A (Sprr1a) and 2A (Sprr1a) were up-regulated by AzaC, but not significantly affected by TCDD. In addition, a group of genes was down-regulated by AzaC, TCDD and their co-treatment. These findings suggest the TCDD-dependent regulation of various genes to be influenced by cellular DNA methylation status.

Animals↗
PubMed2000

Lack of phosphotyrosine phosphatase SHP-1 expression in malignant T-cell lymphoma cells results from methylation of the SHP-1 promoter.

SHP-1 is an important negative regulator of signaling by several receptors including receptors for interleukin-2 (IL-2R) and other cytokines. SHP-1 acts by dephosphorylating the receptors and receptor-associated kinases such as IL-2R-associated Jak3 kinase. We found that SHP-1 protein was not detectable or greatly diminished in most (six of seven) T cell lines derived from various types of T cell lymphomas and all (eight of eight) cutaneous T-cell lymphoma tissues with a transformed, large-cell morphology. All T-cell lymphoma lines tested (eight of eight) expressed diminished amounts or no detectable SHP-1 mRNA. These T cell lines did not, however, carry any mutations in the SHP-1 gene-coding, splice-junction, and promoter regions. Importantly, SHP-1 DNA promoter region in the T cell lines was resistant to digestion with three different methylation-sensitive restriction enzymes. This resistance was reversed by treatment of the cells with a demethylating agent, 5-deoxyazacytidine. The treatment resulted also in the expression of SHP-1 mRNA and, less frequently, SHP-1 protein. The expression of SHP-1 protein was associated with dephosphorylation of the Jak3 kinase. These results show that lack of SHP-1 expression is frequent in malignant T cells and results from methylation of the SHP-1 gene promoter. Furthermore, they indicate that SHP-1 loss may play a role in the pathogenesis of T cell lymphomas by permitting persistence of signals generated by IL-2R and, possibly, other receptor complexes.

Azacitidine↗
PubMed1998

DNA methylation regulates p27kip1 expression in rodent pituitary cell lines.

We previously reported loss of expression of p27Kip1 (p27) protein in rat GH3 and mouse GHRH-CL1 pituitary tumor cells compared with normal pituitary (NP). The molecular basis for the loss of expression of p27 protein in GH3 and GHRH-CL1 cells is unknown. To determine the role of p27 gene methylation in the regulation of the expression of this cell cycle protein, the methylation patterns of p27 in normal and neoplastic pituitary cells was analyzed. Inhibition of DNA methyltransferase (DNA-MTase) with 5-aza-2'-deoxycytidine (AZAdC) induced expression of both p27 protein and mRNA when GH3 and GHRH-CL1 cells were treated for 7 days in vitro. DNA methylation correlated inversely with the expression of p27 gene products in NP and pituitary tumor cell lines. Bisulfite genomic sequencing analysis showed that the normally unmethylated cytosines in exon 1 in NP and AtT20 cells were extensively methylated in GH3 and GHRH-CL1 cells. After treatment of GH3 and GHRH-CL1 cells with 10 micromol/L AZAdC, there were decreased numbers of methylated cytosines (by 60% to 90%/o) with variable methylation patterns observed by bisulfite genomic sequencing. Analysis of genomic DNA with methylation-sensitive enzymes showed that all SmaI, HhaI, and AvaI enzyme sites of the p27 gene in exon 1 were methylated in GH3 cells but not in NP, confirming the bisulfite genomic sequencing results. AtT20 cells and a human pituitary null cell adenoma cell line (HP75), which expressed abundant p27, had a methylation pattern similar to the NP. DNA-MTase activity was elevated fourfold in GH3 cells and twofold in GHRH-CL1 cells compared with DNA-MTase activity in NP and AtT20 cells. These results suggest that increased DNA methylation is another mechanism of silencing of the p27 gene in some pituitary tumors and possibly in other types of neoplasms.

Animals↗
PubMed2003

Detection and analysis of enzymatic DNA methylation of oligonucleotide substrates by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) mass spectrometry was employed to analyze DNA methylation carried out by the Escherichia coli dam DNA methyltransferase using oligonucleotide substrates with molecular masses of 5000-10,000 Da per strand. The mass spectrometry assay offers several advantages: (i) it directly shows the methylation as the increase in the mass of the substrate DNA, (ii) it is nonradioactive, (iii) it is quantitative, and (iv) it can be automated for high-throughput applications. Since unmethylated and methylated DNA are detected, the ratio of methylation can be determined directly and accurately. Furthermore, the assay allows detection individually of the methylation of several substrates in competition, offering an ideal setup to analyze the specificity of DNA interacting with enzymes. We could not identify methylation at any noncanonical site, indicating that the dam MTase is a very specific enzyme. Finally, MALDI-TOF mass spectrometry permitted assessment of the number of methyl groups incorporated into each DNA strand, thereby, allowing study of mechanistic details such as the processivity of the methylation reaction. We provide evidence that the dam MTase modifies DNA in a processive reaction, confirming earlier findings.

DNA Methylation↗