DNA Methyltransferase Inhibitor 5-azacytidine Research Articles

Human concentrative nucleoside transporter 1-mediated uptake of 5-azacytidine enhances DNA demethylation

The DNA methyltransferase inhibitors 5-azacytidine (5-azaCyd) and 5-aza-2'-deoxycytidine have found increasing use for the treatment of myeloid leukemias and solid tumors. Both nucleoside analogues must be transported into cells and phosphorylated before they can be incorporated into DNA and inactivate DNA methyltransferases. The members of the human equilibrative and concentrative nucleoside transporter families mediate transport of natural nucleosides and some nucleoside analogues into cells. However, the molecular identity of the transport proteins responsible for mediating the uptake of 5-azanucleosides has remained unknown. To this end, we have generated a stably transfected Madin-Darby canine kidney strain II cell line expressing recombinant hCNT1. An antiserum directed against hCNT1 specifically detected the protein in the apical membrane of hCNT1-expressing Madin-Darby canine kidney cells. Using [14C]5-azaCyd, we show here that hCNT1 mediated the Na+-dependent uptake of this drug with a Km value of 63 micromol/L. Na+-dependent transport of radiolabeled cytidine, uridine, and 5-fluoro-5'-deoxyuridine further showed the functionality of the transporter. hCNT1-expressing cells were significantly more sensitive to 5-azaCyd, and drug-dependent covalent trapping of DNA methyltransferase 1 was substantially more pronounced. Importantly, these results correlated with a significant sensitization of hCNT1-expressing cells toward the demethylating effects of 5-azaCyd and 5-aza-2'-deoxycytidine. In conclusion, our study identifies 5-azaCyd as a novel substrate for hCNT1 and provides direct evidence that hCNT1 is involved in the DNA-demethylating effects of this drug.

Anti-AML Activity of Combined Epigenetic Therapy with Novel DNMT1 Inhibitors SGI-110 or SGI-1036 and Histone Deacetylase Inhibitor Panobinostat.

Lysine specific histone methylation and deacetylation and DNA hypermethylation are involved in the epigenetic silencing of tumor suppressor genes (TSG), e.g., p15 and p16. DNA methyltransferase (DNMT) inhibitors 5-azacytidine and 5-aza-2‘-deoxycytidine demethylate the CpG dinucleotide islands in or near gene promoters, leading to derepression of TSGs in AML. SGI-110 (S110) (Cancer Res. 2007; 67:6400) and SGI-1036 (SuperGen, Inc.) are novel, DNMT inhibitors, which also deplete DNMT1 levels. SGI-110 is a dinucleotide containing 5-aza-2‘-deoxycytidine and SGI-1036 is a non-nucleoside heterocycle. The multi-protein complex PRC (polycomb repressive complex) 2 that contains the three core proteins EZH2, SUZ12 and EED, has intrinsic histone methyltransferase (HMTase) activity. This is mediated by the SET domain of EZH2, which induces trimethylation of histone H3 on lysine (K)-27. We recently reported that treatment with the pan-HDAC inhibitor panobinostat (LBH589, Novartis Pharmaceutical Corp) acetylates and inhibits the ATP binding and chaperone function of hsp90, as well as depletes the levels of EZH2, Suz12 and EED in cultured and primary AML cells (Mol Cancer Ther. 2006; 5:3096). Within the PRC2 complex, EZH2 was shown to interact with and modulate the DNA methyltransferases DNMT1, DNMT3a and DNMT3b, which affects their binding to the EZH2-targeted gene promoters. In the present studies we determined the effects of SGI-110 or SGI-1036 and LBH589 on the PRC2 proteins EZH2 and SUZ12, and DNMT1, in the cultured (HL-60, OCI-AML3 and K562) and primary AML cells. Treatment with SGI-110 (0.5 to 2.0 μM) or SGI-1036 (0.5 and 1.0 μM) for 24 hours depleted protein levels of DNMT1 and EZH2 in the cultured and primary AML cells. SGI-110 and SGI-1036 promoted proteasomal degradation of DNMT1 and EZH2 since co-treatment with bortezomib significantly restored DNMT1 and EZH2 levels in the AML cells. Following treatment with SGI-110 or SGI-1036, bisulfite modification and methylation specific PCR demonstrated increase in unmethylated promoter DNA of p15 and JunB. This was associated with induction of the mRNA and protein levels of p15 and JunB, as well as caused inhibition of cell cycle progression (% of cells increased in G1 and increased in S phase) and colony growth in the soft agar. Treatment with 1.0 μM of SGI-110 or SGI-1036 also induced PARP cleavage activity of caspases and induced morphologic evidence of apoptosis in the AML cells. Co-treatment with 10 to 50 nM panobinostat enhanced SGI-110 or SGI-1036 mediated depletion of DNMT1 and EZH2, with more de-repression of the p15 and JunB and significant increase in apoptosis of AML cells. Collectively, these findings indicate that, SGI-110 and SGI-1036 deplete DNMT1 and EZH2 levels, as well as exert potent anti-AML activity. Additionally, combined epigenetic therapy consisting of SGI-110 or SGI-1036 in combination with panobinostat may represent a promising novel treatment of AML.

Methylation of the OP-1 promoter: potential role in the age-related decline in OP-1 expression in cartilage

An age-related decline in chondrocyte production of osteogenic protein-1 (OP-1) (Bone Morphogenetic Protein-7) may contribute to cartilage loss in osteoarthritis. This study was designed to determine if increased methylation of the OP-1 promoter might serve as a mechanism for the age-related decline in OP-1 expression. Human articular chondrocytes were isolated from cartilage obtained after death from tissue donors (ages 19-86 years) without a known history of arthritis. DNA was obtained from isolated chondrocytes in primary culture and analyzed for OP-1 promoter methylation by polymerase chain reaction (PCR) after bisulfite treatment. Cultured cells were treated with the DNA methyltransferase inhibitor 5-azacytidine and OP-1 production was measured in the media by enzyme-linked immunosorbent assay (ELISA). RNA was isolated to measure expression of insulin-like growth factor-1 (IGF-1), the IGF-1 receptor (IGF-1R), aggrecan, and OP-1 by real-time PCR. Methylation of the OP-1 promoter was detected in chondrocytes isolated from tissue obtained from older adults and there was a positive correlation between age and OP-1 methylation status (n=22, R(2)=0.277, P=0.014). Inhibition of methylation in cultured cells with 5-azacytidine increased chondrocyte production of OP-1 protein and increased the expression of the IGF-1, the IGF-1R, aggrecan, and OP-1 genes but not GAPDH. Age-related methylation of the OP-1 promoter may contribute to a decrease in OP-1 production in cartilage and a decrease in expression of OP-1 responsive genes such as IGF-1, the IGF-1R, and aggrecan.

Relationships between transcription, silver staining, and chromatin organization of nucleolar organizers in Secale cereale

The nucleolar organizer regions (NORs) are composed of hundreds of rRNA genes, typically spanning several megabases. Cytologically, NORs include regions that are highly condensed and regions that are decondensed, the latter corresponding to regions at which associated proteins stain intensively with silver (Ag-NORs) and where active rRNA gene transcription is thought to occur. To test the relationship between rRNA gene activity, NOR silver staining, and rDNA (genes coding for rRNA) chromatin condensation, we used the DNA methyl-transferase inhibitor 5-azacytidine to evaluate the correlation between the epigenetic regulation of rRNA genes and NOR silver staining in the plant Secale cereale. Following 5-azacytidine treatment, we observed an increase in rRNA gene transcription as well as a reduction in the number of cells showing a significant difference in the size of the silver-stained domains in the two NORs. These transcriptional changes occurred concomitantly with an increase in nuclear and nucleolar size and were associated with the reallocation of most of the rDNA from perinucleolar heterochromatin into the nucleolus. Collectively, these results suggest that rRNA gene transcription, silver staining, and NOR decondensation are interrelated in S. cereale.

Five azacytidine, a DNA methyltransferase inhibitor, specifically inhibits testicular cord formation and Sertoli cell differentiation in vitro

In mammals, Sry, Sox9, and M33 act as regulators at a chromatin level and promote the maturation of embryonic gonads into testes. Recently, it was shown that transcriptional regulation by DNA methylation plays crucial roles in gene expression during the differentiation and development of various cell types. To determine the involvement of DNA methylation in sex determination of the gonad, we developed and performed organ culture of gonad with the DNA methyltransferase inhibitor 5-azacytidine to induce global DNA methylation status changes. In vitro treatment with 5-azacytidine specifically inhibited testicular cord formation in a dose-dependent manner; however, no appreciable defect was observed in ovarian explants. Inhibition of testicular cord was observed only in gonads from 11.5 days post-coitus embryos. These effects were not observed in 5-azacytidine-treated gonads from 12.0 days post-coitus embryos. To determine the effect of 5-azacytidine on Sertoli and Leydig cell differentiation in the testis, we performed whole mount in situ hybridization analysis. The Leydig and stromal cell marker genes Lhx9, Mfge8, and 3beta-Hsd were normally induced in 5-azaytidine-treated testicular explants. Sertoli cell marker genes, Sox9 and MIS were normally induced, but Col9a3, encoding an extracellular matrix component, was inhibited in 5-azacytidine-treated testicular explants. Thus, our data show that DNA methylation is involved in testicular cord formation and Sertoli cell differentiation, acting directly on the gonad at 11.5 days post-coitus.

Myelodysplastic Syndrome (MDS) Displays Profound and Functionally Significant Epigenetic Deregulation Compared to Acute Myeloid Leukemia (AML) and Normal Bone Marrow Cells.

MDS are a clinically heterogeneous group of clonal disorders, which share a high frequency of progression to secondary AML (MDS-AML). In contrast to de novo AML, MDS and MDS-AML are uniformly resistant to conventional chemotherapy. Among the few active drugs in MDS are the nucleoside analog DNA methyltransferase inhibitors (MTIs) 5-azacytidine and decitabine. Since tumor suppressor genes such as CDKN2B can be silenced by DNA methylation in MDS, it is believed that reversal of DNA methylation by MTIs might contribute to their anti-tumor effects. However, it is not clear whether methylation-dependent silencing of specific genes are predictive of response or even correlate with response to MTIs. Like MDS, AML also presents epigenetic silencing of CDKN2B and other genes; yet appear to not be as sensitive to MTIs. Given the particular sensitivity of MDS to MTIs and its resistance to standard AML chemotherapy, we hypothesized that MDS is a biologically distinct disease from AML due largely to extensive epigenetic deregulation, which is missed by single locus studies. In order to test this we studied DNA methylation levels at 24,000 gene promoters in 13 MDS pts. and 16 de novo normal karyotype AML cases, and compared and contrasted these to CD34+ bone marrow cells from 8 healthy donors. For this we used the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) assay, a robust method for detection of whole-genome DNA methylation, and using MassArray quantitative methylation for single locus validation. Remarkably, MDS was found to have a far greater number of hypermethylated genes than AML or normal CD34+ cells, while AML and CD34+ cells had similar number of methylated genes (MDS vs. AML: 6303 vs. 4177 promoters, p=0.026; MDS vs. normal CD34+ cells: 6303 vs. 4296 promoters, p=0.056). Using a moderated T test, an aberrant methylation signature of 736 genes (p<0.0000001) was identified in MDS vs. normal CD34+ cells, reflecting extensive epigenetic deregulation in this disease, including p16, CEBPZ, MSH2, CHES1, AKT1, Caspase2, BMP3, DAP and MYOD1. A comparison between MDS and de novo AML identified 498 genes (p<0.00005) differentially methylated, with a clear predominance of hypermethylated promoters in MDS vs. de novo AML. These genes included RUNX2 and 3, GFI1, DAPK2, MDM2, TGFA, CEBPZ and SHARP. Finally, a comparison between normal CD34+ cells and de novo AML demonstrated an aberrant pattern of methylation in 341 genes (p<0.00001), including CXCL1 and 5, PPARD, Caspase 2 HOXA4 and HOXA10, GFI1 and the MLL translocation partner Septin11. 7 of the 13 MDS cases were also examined for gene expression using the Affymetrix Hgu133plus2 array. A significant proportion of the genes that had been found to be methylated were also found to be underexpressed, including p16, DAP, BMP3, HOXA2 and HOXB8. Taken together, our data show that MDS is a unique and distinct biological entity than de novo AML featuring profound and functionally significant genome wide epigenetic deregulation. While the de novo AML methylation profile was clearly different from normal CD34+ cells, it was not as severely altered as MDS. These data also suggest that MTIs are most likely uniquely active in MDS due to their DNA methyltransferase activity.

RNAi Targeting MBD2 Increases γ-Globin Expression in a CID-Dependent Human β-YAC Murine Fetal Bone Marrow Cell Line.

Increased fetal hemoglobin (HbF) can alleviate the symptoms and increase the life span of patients with sickle cell disease. The development of new methods to increase HbF in patients has been hampered by the lack of cell line models. Recently a new murine fetal bone marrow (FBM) cell line containing the human β-globin gene locus in the context of a yeast artificial chromosome (βYAC) whose growth is dependent on the chemical inducer of dimerization (CID) AP20187 (Ariad Pharmaceuticals) was described (Blau et al, J Biol Chem 280: 36642, 2005). The DNA methyltransferase (DNMTase) inhibitor 5-azacytidine increased γ-globin gene expression in this cell line suggesting that it is a good model system for studying the mechanism of DNA methylation in γ-globin gene silencing. To investigate the role of methylated DNA binding proteins in γ-globin silencing, we transduced this cell line with retrovirus vectors expressing shRNA targeting the methylated DNA binding proteins MBD2 and MBD3. Transduced pools were selected for puromycin resistance and the effect on expression of the target gene and γ-globin determined by real time PCR using the ΔΔCT method. Single cell clones were isolated by limited dilution platings for further analysis. No difference in γ-globin expression was observed between clones derived from the parental cell line and cells transduced with a nonsilencing control vector. MBD3 expression was decreased 70–85% but γ-globin expression was not affected in the selected pool or in 12 clones following transduction wth a vector targeting MBD3. In 2 selected pools transduced with an shRNA vector targeting MBD2, MBD2 expression was decreased 60 and 82% and γ-globin expression increased 3.7 fold and 4.76 fold. Mean expression of γ-globin was 3.83 fold higher in 14 clones that showed >90% decrease in MBD2 mRNA compared to 11 clonal isolates derived from cells transduced with the non-silencing control vector (p<.05). In three clones that showed the greatest induction of γ-globin, the fold change (mean +SD) of ε-, γ-, and β-globin expression was 3.98±1.75, 11.29±4.27 and 1.90±1.34, respectively, demonstrating that MBD2 knockdown increased both ε- and γ-globin expression. The level of MBD2 protein in these clones and in two additional clones that showed >90% decrease in MBD2 mRNA but no induction of γ-globin expression were compared by Western blot analysis. MBD2 was decreased >90% in all clones compared to the parental line. Decreased MBD2 levels were therefore not associated with increased γ-globin expression in all clones. We then compared shRNA-mediated MBD2 knockdown with the DNMTase inhibitor decitabine for effects on ε- and γ-globin expression. Expression of ε-globin was increased 76.0±6.1 fold, γ-globin 131±10.2 fold, and β-globin 3.6±0.38 fold following treatment with decitabine (1 × 10−6 M; 48 hours; n=3). We conclude that decreased expression of MBD2 following transduction of the CID-dependent mouse FBM βYAC cell line with an shRNA vector targeting MBD2 is associated with increased γ-globin expression, confirming previous results of analysis of βYAC-MBD2 knockout mice (Rupon et al, PNAS 103: 6617, 2006). However, the increase in γ-globin expression was approximately 10 fold greater following decitabine treatment than was achieved with shRNA-mediated MBD2 knockdown.

Zebularine reactivates silenced E-cadherin but unlike 5-Azacytidine does not induce switching from latent to lytic Epstein-Barr virus infection in Burkitt's lymphoma Akata cells

Epigenetic silencing of regulatory genes by aberrant methylation contributes to tumorigenesis. DNA methyltransferase inhibitors (DNMTI) represent promising new drugs for anti-cancer therapies. The DNMTI 5-Azacytidine is effective against myelodysplastic syndrome, but induces switching of latent to lytic Epstein-Barr virus (EBV) in vitro and results in EBV DNA demethylation with the potential of induction of lytic EBV in vivo. This is of considerable concern given that recurrent lytic EBV has been linked with an increased incidence of EBV-associated lymphomas. Based on the distinct properties of action we hypothesized that the newer DNMTI Zebularine might differ from 5-Azacytidine in its potential to induce switching from latent to lytic EBV. Here we show that both 5-Azacytidine and Zebularine are able to induce expression of E-cadherin, a cellular gene frequently silenced by hypermethylation in cancers, and thus demonstrate that both DNMTI are active in our experimental setting consisting of EBV-harboring Burkitt's lymphoma Akata cells. Quantification of mRNA expression of EBV genes revealed that 5-Azacytidine induces switching from latent to lytic EBV and, in addition, that the immediate-early lytic infection progresses to early and late lytic infection. Furthermore, 5-Azacytidine induced upregulation of the latent EBV genes LMP2A, LMP2B, and EBNA2 in a similar fashion as observed following switching of latent to lytic EBV upon cross-linking of the B-cell receptor. In striking contrast, Zebularine did not exhibit any effect neither on lytic nor on latent EBV gene expression. Thus, Zebularine might be safer than 5-Azacytidine for the treatment of cancers in EBV carriers and could also be applied against EBV-harboring tumors, since it does not induce switching from latent to lytic EBV which may result in secondary EBV-associated malignancies.

5-Azacytidine induces changes in electrophysiological properties of human mesenchymal stem cells

Previously, mouse bone marrow-derived stem cells (MSC) treated with the unspecific DNA methyltransferase inhibitor 5-azacytidine were reported to differentiate into cardiomyocytes. The aim of the present study was to investigate the efficiency of a similar differentiation strategy in human mononuclear cells obtained from healthy bone marrow donors. After 1-3 passages, cultures were exposed for 24 h to 5-azacytidine (3 mciroM) followed by 6 weeks of further culture. Drug treatment did not induce expression of myogenic marker MyoD or cardiac markers Nkx2.5 and GATA-4 and did not yield beating cells during follow-up. In patch clamp experiments, approximately 10-15% of treated and untreated cells exhibited L-type Ca(2+) currents. Almost all cells showed outwardly rectifying K(+) currents of rapid or slow activation kinetics. Mean current amplitude at +60 mV doubled after 6 weeks of treatment compared with time-matched controls. Membrane capacitance of treated cells was significantly larger than in controls 2 weeks after treatment and remained high after 6 weeks. Expression levels of mRNAs for the K(+) channels Kv1.1, Kv1.5, Kv2.1, Kv4.3 and KCNMA1 and for the Ca(2+) channel Ca(v)1.2 were not affected by 5-azacytidine. Treatment with potassium channel blockers tetraethylammonium and clofilium at concentrations shown previously to inhibit rapid or slowly activating K(+) currents of hMSC inhibited proliferation of these cells. Our results suggest that despite the absence of differentiation of hMSC into cardiomyocytes, treatment with 5-azacytidine caused profound changes in current density.

MUC2 expression is regulated by histone H3 modification and DNA methylation in pancreatic cancer

Mucins are highly glycosylated proteins that play important roles in carcinogenesis. In pancreatic neoplasia, MUC2 mucin has been demonstrated as a tumor suppressor and we have reported that MUC2 is a favorable prognostic factor. Regulation of MUC2 gene expression is known to be controlled by DNA methylation, but the role of histone modification for MUC2 gene expression has yet to be clarified. Herein, we provide the first report that the histone H3 modification of the MUC2 promoter region regulates MUC2 gene expression. To investigate the histone modification and DNA methylation of the promoter region of the MUC2 gene, we treated 2 human pancreatic cancer cell lines, PANC1 (MUC2-negative) and BxPC3 (MUC2-positive) with the DNA methyltransferase inhibitor 5-azacytidine (5-aza), the histone deacetylase inhibitor trichostatin A (TSA), and a combination of these agents. The DNA methylation level of PANC1 cells was decreased by all 3 treatments, whereas histone H3-K4/K9 methylation and H3-K9/K27 acetylation in PANC1 cells was changed to the level in BxPC3 cells by treatment with TSA alone and with the 5-aza/TSA combination. The expression level of MUC2 mRNA in PANC1 cells exhibited a definite increase when treated with TSA and 5-aza/TSA, whereas 5-aza alone induced only a slight increase. Our results suggest that histone H3 modification in the 5' flanking region play an important role in MUC2 gene expression, possibly affecting DNA methylation. An understanding of these intimately correlated epigenetic changes may be of importance for predicting the outcome of patients with pancreatic neoplasms.

Methyl binding domain protein 2 mediates γ-globin gene silencing in adult human βYAC transgenic mice

The genes of the vertebrate beta-globin locus undergo a switch in expression during erythroid development whereby embryonic/fetal genes of the cluster are sequentially silenced and adult genes are activated. We describe here a role for DNA methylation and MBD2 in the silencing of the human fetal gamma-globin gene. The gamma-globin gene is reactivated upon treatment with the DNA methyltransferase inhibitor 5-azacytidine in the context of a mouse containing the entire human beta-globin locus as a yeast artificial chromosome (betaYAC) transgene. To elucidate the mechanism through which DNA methylation represses the gamma-globin gene in adult erythroid cells, betaYAC/MBD2-/- mice were generated by breeding betaYAC mice with MBD2-/- mice. Adult betaYAC/MBD2-/- mice continue to express the gamma-globin gene at a level commensurate with 5-azacytidine treatment, 10- to 20-fold over that observed with 1-acetyl-2-phenylhydrazine treatment alone. In addition, the level of gamma-globin expression is consistently higher in MBD2-/- mice in 14.5- and 16.5-days postcoitus fetal liver erythroblasts suggesting a role for MBD2 in embryonic/fetal erythroid development. DNA methylation levels are modestly decreased in MBD2-/- mice. MBD2 does not bind to the gamma-globin promoter region to maintain gamma-globin silencing. Finally, treatment of MBD2-null mice with 5-azacytidine induces only a small, nonadditive induction of gamma-globin mRNA, signifying that DNA methylation acts primarily through MBD2 to maintain gamma-globin suppression in adult erythroid cells.

LUMA (LUminometric Methylation Assay)—A high throughput method to the analysis of genomic DNA methylation

Changes in genomic DNA methylation are recognized as important events in normal and pathological cellular processes, contributing both to normal development and differentiation as well as cancer and other diseases. Here, we report a novel method to estimate genome-wide DNA methylation, referred to as LUminometric Methylation Assay (LUMA). The method is based on combined DNA cleavage by methylation-sensitive restriction enzymes and polymerase extension assay by Pyrosequencing™. The method is quantitative, highly reproducible and easy to scale up. Since no primary modification of genomic DNA, such as bisulfite treatment, is needed, the total assay time is only 6 h. In addition, the assay requires only 200–500 ng of genomic DNA and incorporates an internal control to eliminate the problem of varying amounts of starting DNA. The accuracy and linearity of LUMA were verified by in vitro methylated lambda DNA. In addition, DNA methylation levels were assessed by LUMA in DNA methyltransferase knock-out cell lines and after treatment with the DNA methyltransferase inhibitor (5-AzaCytidine). The LUMA assay may provide a useful method to analyze genome-wide DNA methylation for a variety of physiological and pathological conditions including etiologic, diagnostic and prognostic aspects of cancer.

Oral Administration of Dacogen Mesylate Increases HbF and Decreases DNA Methylation of the ε- and γ-Globin Genes in Anemic Baboons (Papio anubis).

Increased levels of fetal hemoglobin (HbF) lessen the severity of sickle cell disease. Subcutaneous and intravenous adminstration of a drug that inhibits DNA methyltransferase, 5-aza-2′-deoxycytidine (Dacogen; DAC), increased HbF levels in hydroxyurea-refractory sickle cell patients and experimentally-induced anemic baboons. An orally administered, effective DNA methyltransferase inhibitor would be desirable for increasing compliance of patients in future clinical trials. Previous experiments showed that oral administration of the DNA methyltransferase inhibitor 5-azacytidine was only effective when combined with tetrahydrouridine, a cytidine deaminase inhibitor. Zebularine inhibits DNA methyltransferase when administered orally, however relatively high doses are required and the ability of this drug to increase HbF levels has not been tested in either experimental primate studies or in patients with sickle cell disease. Based upon previous studies in mice demonstrating bioavailability of orally administered DAC, we tested whether a new salt form, DAC mesylate, could increase HbF and cause DNA demethylation when administered orally at doses 8–36 fold higher than effective doses given subcutaneously. Three baboons were rendered anemic by acute phlebotomy for ten days and maintained at a Hct of 20 during the course of drug treatment. HbF levels following the initial bleeding and prior to DAC mesylate administration were 6.3–13.9%. Each baboon received a different orally administered dose of DAC mesylate (18.7mg/kg/day; 9.35mg/kg/day; 4.1mg/kg/day) for ten days. Peak HbF levels achieved in animals receiving these three different doses were 67.8, 61.9, and 17.4, respectively. Peak HbF in the two animals receiving higher doses were comparable to levels observed in these animals following subcutaneous injection of a lower dose of DAC (0.52mg/kg/day). Bisulfite sequence analysis showed that methylation of the ε- and γ-globin genes was decreased >50% in animals treated with 18.7mg/kg and 9.35mg/kg doses, while minimal changes were observed in the animal treated with the lowest dose (4.1mg/kg). Chromatin immunoprecipitation (ChIP) studies showed that the levels of acetylated histones H3 and H4 associated with the β-globin promoter were 5–6 fold higher than with the γ-globin promoter in bled animals. Following DAC mesylate, equivalent levels of acetylated histones H3 and H4 were associated with the γ- and β-globin promoters in the two animals treated with the higher doses of drug. These studies thus demonstrate that orally administered DAC mesylate increased HbF, reduced DNA methylation of the ε- and γ-globin genes, and increased acetylation of histones H3 and H4 associated with the γ-globin promoter in anemic baboons. Future experiments to directly compare the efficacy of DAC mesylate and DAC in baboons and potential Phase I studies to assess the feasibility of oral administration of DAC mesylate in the treatment of patients with sickle cell disease may be warranted.Effect of Oral DAC Mesylate on HbF and DNA MethylationAnimalTreatmentDAC Dose (mg/kg/day)ε-globin (% dmC)γ-globin (% dmC)HbF (%)PA 6974Bled096.679.36.3Oral DAC mesylate18.743.335.067.8PA 7002Bled086.671.713.9Oral DAC mesylate9.3546.63461.9PA 7001Bled090.078.76.3Oral DAC mesylate4.186.674.117.4

Epigenetic Basis for the Transcriptional Hyporesponsiveness of the Human Inducible Nitric Oxide Synthase Gene in Vascular Endothelial Cells

A marked difference exists in the inducibility of inducible NO synthase (iNOS) between humans and rodents. Although important cis and trans factors in the murine and human iNOS promoters have been characterized using episomal-based approaches, a compelling molecular explanation for why human iNOS is resistant to induction has not been reported. In this study we present evidence that the hyporesponsiveness of the human iNOS promoter is based in part on epigenetic silencing, specifically hypermethylation of CpG dinucleotides and histone H3 lysine 9 methylation. Using bisulfite sequencing, we demonstrated that the iNOS promoter was heavily methylated at CpG dinucleotides in a variety of primary human endothelial cells and vascular smooth muscle cells, all of which are notoriously resistant to iNOS induction. In contrast, in human cell types capable of iNOS induction (e.g., A549 pulmonary adenocarcinoma, DLD-1 colon adenocarcinoma, and primary hepatocytes), the iNOS promoter was relatively hypomethylated. Treatment of human cells, such as DLD-1, with a DNA methyltransferase inhibitor (5-azacytidine) induced global and iNOS promoter DNA hypomethylation. Importantly, 5-azacytidine enhanced the cytokine inducibility of iNOS. Using chromatin immunoprecipitation, we found that the human iNOS promoter was basally enriched with di- and trimethylation of H3 lysine 9 in endothelial cells, and this did not change with cytokine addition. This contrasted with the absence of lysine 9 methylation in inducible cell types. Importantly, chromatin immunoprecipitation demonstrated the selective presence of the methyl-CpG-binding transcriptional repressor MeCP2 at the iNOS promoter in endothelial cells. Collectively, our work defines a role for chromatin-based mechanisms in the control of human iNOS gene expression.

Overexpression of CD70 and overstimulation of IgG synthesis by lupus T cells and T cells treated with DNA methylation inhibitors.

Generalized DNA hypomethylation contributes to altered T cell function and gene expression in systemic lupus erythematosus (SLE). Some of the overexpressed genes participate in the disease process, but the full repertoire of genes affected is unknown. Methylation-sensitive T cell genes were identified by treating T cells with the DNA methyltransferase inhibitor 5-azacytidine and comparing gene expression with oligonucleotide arrays. CD70, a costimulatory ligand for B cell CD27, was one gene that reproducibly increased. We then determined whether CD70 is overexpressed on T cells treated with other DNA methylation inhibitors and on SLE T cells, and determined its functional significance. Oligonucleotide arrays, real-time reverse transcription-polymerase chain reaction, and flow cytometry were used to compare CD70 expression in T cells treated with 2 DNA methyltransferase inhibitors (5-azacytidine and procainamide) and 3 ERK pathway inhibitors known to decrease DNA methyltransferase expression (U0126, PD98059, and hydralazine). The consequences of CD70 overexpression were tested by coculture of autologous T and B cells with and without anti-CD70 and measuring IgG production by enzyme-linked immunosorbent assay. The results were compared with those of T cells from lupus patients. SLE T cells and T cells treated with DNA methylation inhibitors overexpressed CD70 and overstimulated B cell IgG production. The increase in IgG synthesis was abrogated by anti-CD70. SLE T cells and T cells treated with DNA methyltransferase inhibitors and ERK pathway inhibitors overexpress CD70. This increased B cell costimulation and subsequent immunoglobulin overproduction may contribute to drug-induced and idiopathic lupus.

MDR1 Promoter Hypermethylation in MCF-7 Human Breast Cancer Cells: Changes in Chromatin Structure Induced by Treatment with 5-Aza-Cytidine

Resistance to the cytotoxic actions of antineoplastic drugs, whether intrinsic or acquired, remains a barrier to the establishment of curative chemotherapy regimens for advanced breast cancer. Over-expression of P-glycoprotein (P-gp), encoded by the MDR1 gene and known to mediate resistance to many antineoplastic drugs, may contribute to poor breast cancer treatment outcome. Nonetheless, the precise molecular mechanisms responsible for high or low level P-gp expression in breast cancer cells have not been established. We assessed the role of DNA hypermethylation near the MDR1 transcriptional regulatory region in MDR1 expression in MCF-7 breast cancer cells, which fail to express MDR1 mRNA, and MCF-7/ADR cells, known to express high MDR1 mRNA levels. When compared to MCF-7/ADR cells, MCF-7 cells manifested markedly diminished MDR1 transcription rates by nuclear run-off assay, but equivalent MDR1 promoter trans-activation activity in transient transfection experiments, indicating that cis factors were most likely responsible for the differences in MDR1 transcription between MCF-7/ADR cells and MCF-7 cells. Bisulfite genomic sequencing analyses revealed substantially less extensive MDR1 promoter methylation in MCF-7/ADR cells than in MCF-7 cells, suggesting that CpG dinucleotide methylation might contribute to the observed MDR1 transcription differences. Chromatin immunoprecipitation analyses indicated an inactive MDR1 chromatin conformation in MCF-7 cells, with a paucity of acetylated histones and the presence of 5-mC-binding proteins MeCP2 and MBD2, and an active MDR1 chromatin conformation in MCF-7/ADR cells, with an abundance of acetylated histones and the presence of the transcriptional trans-activator YB-1. Stable MCF-7 sublines which had been treated with the DNA methyltransferase inhibitor 5-azacytidine, exhibited a reduction in MDR1 promoter methylation and a complex MDR1 chromatin configuration, characterized by the simultaneous presence of transcriptional activators and repressors. In this state, MDR1 expression was markedly sensitive to treatment with the histone deacetylase inhibitor trichostatin A.

Analysis of specific lysine histone H3 and H4 acetylation and methylation status in clones of cells with a gene silenced by nickel exposure

We have previously reported that the gpt transgene in G12 Chinese hamster cells could be silenced by water-insoluble nickel compounds nickel sulfide (NiS) or nickel subsulfide (Ni 3S 2) and showed that the transgene was silenced by de novo DNA methylation and chromatin condensation. To further understand the nature of this silencing, we used the chromatin immunoprecipitation assay to elucidate the chromatin structure in nickel-induced silenced G12 clones. We also analyzed the effects of the DNA methyltransferase inhibitor 5-azacytidine (5-AzaC) and a histone deacetylase inhibitor trichostatin A (TSA) on histone H3 and H4 acetylation and gpt gene expression in selected nickel-silenced clones. We observed that both histone H3 and H4 were hypoacetylated and a methyl DNA-binding protein MeCP2 was targeted to the gpt gene locus, resulting in a localized inactive chromatin configuration in nickel-silenced cell clones. The histone H3K9 was also found methylated in three of four nickel- silenced cell clones, whereas the histone H3K9 was deacetylated in all four cell clones, indicating that the H3K9 methylation was involved in nickel-induced gene silencing. The acetylation of the gpt gene could be increased by a combination of 5-AzaC and TSA treatment, but not by either 5-AzaC or TSA alone. The gpt transcript was studied by either Northern blot or by semiquantitative RT–PCR following treatment of the silenced clones with TSA or 5-AzaC. An increase in gpt mRNA could be detected by RT–PCR in the clones that regained acetylation of H3 and H4. These data show that gene silencing induced by nickel in the gpt transgenic cell line involved a loss of histone acetylation and an activation of histone methylation. Both H4 and H3 histone acetylation were lost in the silenced clones and these clones exhibited an increase in the methylation of the lysine 9 in histone H3.

Establishment and functional validation of a structural homology model for human DNA methyltransferase 1

Changes in DNA methylation patterns play an important role in tumorigenesis. The DNA methyltransferase 1 (DNMT1) protein represents a major DNA methyltransferase activity in human cells and is therefore a prominent target for experimental cancer therapies. However, there are only few available inhibitors and their high toxicity and low specificity have so far precluded their broad use in chemotherapy. Based on the strong conservation of catalytic DNA methyltransferase domains we have used a homology modeling approach to determine the three-dimensional structure of the DNMT1 catalytic domain. Our results suggest an overall structural conservation with other DNA methyltransferases but also indicate local conformational differences. To prove the validity of our model we used it as a template to design a novel derivative of the known DNA methyltransferase inhibitor 5-azacytidine. The resulting compound (N4-fluoroacetyl-5-azacytidine) functioned as an efficient inhibitor of DNA methylation in human tumor cell lines and also provides novel opportunities for pharmacological applications.

DNA methylation and chromatin structure regulate T cell perforin gene expression.

Perforin is a cytotoxic effector molecule expressed in NK cells and a subset of T cells. The mechanisms regulating its expression are incompletely understood. We observed that DNA methylation inhibition could increase perforin expression in T cells, so we examined the methylation pattern and chromatin structure of the human perforin promoter and upstream enhancer in primary CD4(+) and CD8(+) T cells as well as in an NK cell line that expresses perforin, compared with fibroblasts, which do not express perforin. The entire region was nearly completely unmethylated in the NK cell line and largely methylated in fibroblasts. In contrast, only the core promoter was constitutively unmethylated in primary CD4(+) and CD8(+) cells, and expression was associated with hypomethylation of an area residing between the upstream enhancer at -1 kb and the distal promoter at -0.3 kb. Treating T cells with the DNA methyltransferase inhibitor 5-azacytidine selectively demethylated this area and increased perforin expression. Selective methylation of this region suppressed promoter function in transfection assays. Finally, perforin expression and hypomethylation were associated with localized sensitivity of the 5' flank to DNase I digestion, indicating an accessible configuration. These results indicate that DNA methylation and chromatin structure participate in the regulation of perforin expression in T cells.

Reconstitution of TIMP-2 expression in SH-SY5Y neuroblastoma cells by 5-azacytidine is mediated transcriptionally by NF-Y through an inverted CCAAT site

Advanced stage neuroblastomas (NB) exhibit a tissue inhibitor of metalloproteinase (TIMP)-2/matrix metalloproteinase (MMP) imbalance, considered a prerequisite for MMP involvement in tumor progression in vivo. Human SH-SY5Y NB cells exhibit a similar TIMP-2/MMP imbalance that promotes in vitro invasive behavior that is inhibited by exogenous TIMP-2. The DNA methyltransferase inhibitor 5-azacytidine (5-AzaC) redresses this TIMP-2/MMP imbalance, reconstituting TIMP-2 expression, without effecting that of MMP-2, by stimulating TIMP-2 transcription and inhibiting in vitro invasivity of SH-SY5Y cells. 5-AzaC stimulated transcription from a nonmethylated TIMP-2 promoter reporter gene construct consistent with regulation of a TIMP-2 transactivator. Promoter deletion and point-mutation analysis localized this effect to an inverted CCAAT element at position −73. This element bound specific complexes containing NF-YA and NF-YB proteins in SH-SY5Y nuclear extracts, the binding of which was augmented by 5-AzaC in association with enhanced levels of NF-YB protein and the function of which was confirmed by inhibition using dominant-negative NF-YA. The data highlight a novel indirect methylation-mediated mechanism for regulating the TIMP/MMP equilibrium in NB cells, involving repression of TIMP-2 relative to MMP-2 expression, dependent upon suboptimal NF-Y transcription factor function, which can be reversed by methyltransferase inhibition.