DNMT1 Protein Levels Research Articles

Abstract 3796: Inhibition of epigenetic chromatin-modification enzymes: histone deacetylases and DNA methyltransferases by silibinin in human NSCLC H1299 cells

Abstract Epigenetic modifications of histones (e.g. hypoacetylation) and DNA (e.g. hypermethylation) cause reversible alterations in chromatin structure leading to aberrant changes in gene expression, which eventually impact cellular processes like cell cycle, apoptosis, and invasion during malignant transformation of cells. Several tumor suppressor genes like CDH1, RARB, RASSF1A, MGMT etc are silenced by these interlinked mechanisms in human lung cancer; the leading diagnosed and lethal malignancy world wide. Hence, the impetus has been on developing novel agents that target ‘epigenome’ for both cancer therapy and chemoprevention. Silibinin, a flavanolignan isolated from the seeds of milk thistle exerts strong anti-cancer efficacy against many epithelial cancers including lung. Recently, we showed that silibinin inhibits p53 null-H1299 cell growth through G1 arrest by modulating the expression and function of associated key cell-cycle regulators. Herein, we assessed the effect of silibinin on major chromatin-modification enzymes to delineate their possible involvement in the observed silibinin effects. A time response study for histone deacetylase (HDAC) activity in total cell extract showed that silibinin treatment causes a dose-dependent decrease in enzyme activity from 6h [(100 µM; p<0.05)]-72h [(75-100 µM; p<0.001)]. Concomitantly, under similar treatment conditions, a sustained accumulation of acetylated histones (H3 and H4) in total cellular chromatin was observed between 6-24h time points. We also addressed the question whether silibinin directly inhibits HDAC activity in a cell free system. In both total and nuclear cell extracts, a decreasing trend in enzyme activity was observed by silibinin addition in incubation reactions, but the concentration for 50% inhibition (IC50) was ∼ 400 µM, suggesting an indirect effect of silibinin in regulating HDAC activity in cell free system. Consistent with these results, silibinin (75-100 µM) also caused a decrease in the protein levels of HDAC1, HDAC2, HDAC3, DNMT1 and DNMT3A in nuclear and total extracts after 24, 48 and 72h of treatment. Next, we evaluated the expression profile of 84 key genes encoding chromatin-modification enzymes in H1299 cells after silibinin treatment (100 µM; 72h). Real time RT-PCR array analysis identified a subset of fifteen genes, primarily including DNMT3A, HDAC1, HDAC6, SET domain proteins (SETD1A, D4, D6) and lysine specific demethylases (KDM 5B, 5C, 4A) which were significantly down-regulated (∼2-4 fold) in silibinin treated samples compared to vehicle controls. Collectively, these results show that silibinin inhibits specific histone deacetylases, histone demethylases and DNA methyltransferases, which might cooperatively contribute to the anti-cancer efficacy of this non-toxic phytochemical. (Supported by CA113876) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3796.

Abstract 5364: Zeranol Down-regulates p53 Expression in Primary Cultured Human Breast Cancer Epithelial Cells through Epigenetic Modification

Abstract Despite great improvements in diagnosis, treatment advances, and healthier life-styles, breast cancer remains the most common cancer among women in the United States. It is estimated that 192,370 new cases arise and 40,170 women will die from breast cancer in 2009. Epidemiological studies have suggested that there are many risk factors associated with breast cancer such as dietary fat and environmental estrogenic endocrine disruptors. Zeranol (Z), as one of the six growth promoters approved by FDA, has been widely used to enhance the growth of beef cattle. Researchers are still in disagreement whether the growth promoters pose any adverse health risk from consuming beef products produced from Z-implanted beef cattle. Our previous results showed that pre-adipocytes isolated from the cattle 60 days post Z-implantation (72 mg/pellet/animal) grow about 12 fold faster than the pre-adipocytes isolated from the control cattle. Treatment with 0.2, 1 and 5% sera (Z-Sera) harvested from the cattle 30 days post implantation in culture medium significantly stimulated MCF-10A, a human normal breast epithelial cells, and MCF-7 cell growth. Proliferation might be caused by Z-Sera down-regulating the expression of a tumor suppressor gene, protein tyrosine phosphatase γ (PTP γ). The current study investigated the effects of Z on primary cultured human normal and cancerous breast epithelial cell growth (PCHNBECs, PCHBCECs), and the regulation of p53 expression, as well as the potential mechanism. Using non-radioactive cell proliferation assay, real time PCR and western blot analysis, we demonstrated that PCHBCECs are more sensitive in response to treatment with Z than PCHNBECs. Treatment with 7.5, 15 and 30 nM Z increased the growth of PCHNBECs at 27, 45, 50%, respectively, as compared to the control group. The same concentrations enhanced the proliferation of PCHBCECs at 25, 84 and 91%. The expression of p53 protein in PCHNBECs did not decrease by the treatment of Z. However, in PCHBCECs, treatment with 7.5, 15 and 30 nM Z significantly down-regulated the expression of p53 protein by 10, 20 and 40%, respectively. Further investigation suggests that the effect of Z on decreasing the expression of p53 in PCHBCECs might be mediated through its epigenetic modification. Our results showed that Z treatment up-regulates the expression of DNMT1 mRNA by 36, 58 and 160%, and protein by 14, 25 and 42% in PCHBCECs after 24 hour exposure. Our data suggest that the stimulatory effect of Z on PCHBCECs growth might be via down-regulation of p53 expression as a result of epigenetic modification mediated through Z enhancing the expression of DNMT1 mRNA and protein levels. These results imply the potential adverse health risks of the consumption of beef products with Z or its bio-active metabolites. Further investigation on this critical issue is in-progress in our laboratory (Supported by NIH grant R01 ES015212-01). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5364.

241 GENE EXPRESSION OF DNMT1 ISOFORMS IN PORCINE OOCYTES, EMBRYOS, AND SOMATIC CELLS

In human, mouse, and some marsupials, the dynamics of genomic methylation and the initial events of gametic imprinting are controlled by the activity of an oocyte isoform of the DNA methyltransferase-1 (DNMT1o) enzyme. The identification and characterization of a similar oocyte transcript variant in farm animals would greatly contribute to the understanding of the methylation processes that occur during nuclear remodeling of in vivo embryos as well as in cloned embryos. The objectives of this study were to identify the alternative splicing variants of DNMT1 in porcine oocytes and determine the gene expression pattern of the different DNMT1 isoforms during embryo development. Total RNA was isolated from a pool of 50 zona free mature porcine oocytes using the Trizol method. A RACE System (Invitrogen, Carlsbad, CA) was used to amplify the 5′ cDNA end of DNMT1. Polymerase chain reaction products were separated by electrophoresis, recovered from the agarose gel, subcloned into a cloning vector, and sequenced. The location of exons and introns of every transcript variant was determined by aligning the 5′RACE-derived sequences to the Sus scrofa DNMT1 genomic sequence located on chromosome 2 (CU462940). RNA levels of the DNMT1 isoforms were analyzed in porcine oocytes and in vivo embryos, as well and in cells from ovary, lung, spleen, liver, heart, and skin by quantitative PCR using the AACT method. DNMT1 protein levels of porcine oocytes and different somatic cells types were analyzed by Western blot. Two new DNMT1o RNA isoforms were identified (EU908730 and EU908731). The previously reported DNMT1s isoform (DQ060156) was expressed at low but constant levels in oocytes, as well as in embryos from the 2-cell to the blastocyst stage. Abundant RNA levels of EU908730 and EU908731 were detected in oocytes and embryos from the 2-cell to the 8- to 16-cell stage. Levels of these DNMT1o transcripts were low at the morula and blastocyst stage. Although DQ060156 was present in all the somatic cell types analyzed, EU908730 and EU908731 were not detected in any somatic tissues. As predicted by the RNA sequence and verified by Western blot analysis, EU908730 and EU908731 RNAs translate one DNMT1o enzyme (ˆ170 KDa). Western blot analysis confirmed that both the oocyte and the somatic forms of DNMT1 protein are present in porcine oocytes and early embryos, while somatic cells produce only DNMT1s protein. In conclusion, this study demonstrates that porcine oocytes express a DNMT1o RNA in addition to the DNMT1s somatic transcript. The 2 newly identified RNA isoforms are produced by alternative splicing of the DNMT1 gene and translate a DNMT1o protein that is unique to oocytes and early embryos. Analysis of the oocyte and somatic DNMT1 isoforms in pre-implantation embryos will determine the expression pattern of this transcript during genomic methylation and its involvement during nuclear reprogramming and cellular differentiation.

Panobinostat treatment depletes EZH2 and DNMT1 levels and enhances decitabine mediated de-repression of JunB and loss of survival of human acute leukemia cells

The PRC2 complex protein EZH2 is a histone methyltransferase that is known to bind and recruit DNMT1 to the DNA to modulate DNA methylation. Here, we determined that the pan-HDAC inhibitor panobinostat (LBH589) treatment depletes DNMT1 and EZH2 protein levels, disrupts the interaction of DNMT1 with EZH2, as well as de-represses JunB in human acute leukemia cells. Similar to treatment with the hsp90 inhibitor 17-DMAG, treatment with panobinostat also inhibited the chaperone association of heat shock protein 90 with DNMT1 and EZH2, which promoted the proteasomal degradation of DNMT1 and EZH2. Unlike treatment with the DNA methyltransferase inhibitor decitabine, which demethylates JunB promoter DNA, panobinostat treatment mediated chromatin alterations in the JunB promoter. Combined treatment with panobinostat and decitabine caused greater attenuation of DNMT1 and EZH2 levels than either agent alone, which was accompanied by more JunB de-repression and loss of clonogenic survival of K562 cells. Co-treatment with panobinostat and decitabine also caused more loss of viability of primary AML but not normal CD34+ bone marrow progenitor cells. Collectively, these findings indicate that co-treatment with panobinostat and decitabine targets multiple epigenetic mechanisms to de-repress JunB and exerts antileukemia activity against human acute myeloid leukemia cells.

DNA Methyltransferase Protein Synthesis Is Reduced in CXXC Finger Protein 1–Deficient Embryonic Stem Cells

CXXC finger protein 1 (CFP1) binds to unmethylated CpG dinucleotides and is required for embryogenesis. CFP1 is also a component of the Setd1A and Setd1B histone H3K4 methyltransferase complexes. Murine embryonic stem (ES) cells lacking CFP1 fail to differentiate, and exhibit a 70% reduction in global genomic cytosine methylation and a 50% reduction in DNA methyltransferase (DNMT1) protein and activity. This study investigated the underlying mechanism for reduced DNMT1 expression in CFP1-deficient ES cells. DNMT1 transcript levels were significantly elevated in ES cells lacking CFP1, despite the observed reduction in DNMT1 protein levels. To address the posttranscriptional mechanisms by which CFP1 regulates DNMT1 protein activity, pulse/chase analyses were carried out, demonstrating a modest reduction in DNMT1 protein half-life in CFP1-deficient ES cells. Additionally, global protein synthesis was decreased in ES cells lacking CFP1, contributing to a reduction in the synthesis of DNMT1 protein. ES cells lacking CFP1 were found to contain elevated levels of phosphorylated eIF2alpha, and an accompanying reduction in translation initiation as revealed by a lower level of polyribosomes. These results reveal a novel role for CFP1 in the regulation of translation initiation, and indicate that loss of CFP1 function leads to decreased DNMT1 protein synthesis and half-life.

Alternations of DNA methylation pattern by post-weaning social isolation in frontal cortex of Fischer 344 rats

The present work was designed to study the mechanisms associated with Nurr1 modulation following early life permethrin (PERM) treatment during rat’s life span. Here we demonstrate that PERM exposure in rats, at a dose close to No Observed Adverse Effect Level (NOAEL) for 15 days during neonatal brain development leads to its accumulation long after exposure. In striatum from adolescent rats we detected an increase in DNA methyltransferases (DNMTs) such as DNMT1, DNMT3a, Tyrosine hydroxylase, monomeric and aggregated α-synuclein protein levels. Adult rats showed enhanced DNMT3b and α-synuclein aggregation compared to the control group, while with aging a significant decrease in all biomarkers studied was observed. No changes in Nurr1 promoter methylation in adolescent, adult and old rats were found.In silico studies showed clear evidence of a strong binding interaction between PERM and its metabolite 3-phenoxybenzoic acid with the nuclear orphan receptor Nurr1. These findings suggest that an additional interference with the dopaminergic neuron pathway could occur in situ during PERM accumulation in brain.Therefore, Nurr1 modulation in early life PERM-treated rats, depends on age-related adaptive responses in animals.

The Reelin and GAD67 Promoters Are Activated by Epigenetic Drugs That Facilitate the Disruption of Local Repressor Complexes

The epigenetic down-regulation of genes is emerging as a possible underlying mechanism of the GABAergic neuron dysfunction in schizophrenia. For example, evidence has been presented to show that the promoters associated with reelin and GAD67 are down-regulated as a consequence of DNA methyltransferase (DNMT)-mediated hypermethylation. Using neuronal progenitor cells to study this regulation, we have previously demonstrated that DNMT inhibitors coordinately increase reelin and GAD67 mRNAs. Here, we report that another group of epigenetic drugs, histone deacetylase (HDAC) inhibitors, activate these two genes with dose and time dependence comparable with that of DNMT inhibitors. In parallel, both groups of drugs decrease DNMT1, DNMT3A, and DNMT3B protein levels and reduce DNMT enzyme activity. Furthermore, induction of the reelin and GAD67 mRNAs is accompanied by the dissociation of repressor complexes containing all three DNMTs, MeCP2, and HDAC1 from the corresponding promoters and by increased local histone acetylation. Our data imply that drug-induced promoter demethylation is relevant for maximal activation of reelin and GAD67 transcription. The results suggest that HDAC and DNMT inhibitors activate reelin and GAD67 expression through similar mechanisms. Both classes of drugs attenuate, directly or indirectly, the enzymatic and transcriptional repressor activities of DNMTs and HDACs. These data provide a mechanistic rationale for the use of epigenetic drugs, individually or in combination, as a potential novel therapeutic strategy to alleviate deficits associated with schizophrenia.

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.

CFP1 interacts with DNMT1 independently of association with the Setd1 Histone H3K4 methyltransferase complexes.

CXXC finger protein 1 (CFP1) is a component of the Setd1A and Setd1B methyltransferase complexes, localizes to euchromatic regions of the genome, and specifically binds unmethylated CpG dinucleotides in DNA. Murine embryos lacking CFP1 exhibit peri-implantation lethality, a developmental time that correlates with global epigenetic reprogramming. CFP1-deficient embryonic stem (ES) cells exhibit a 70% reduction in global cytosine methylation and a 60% decrease in maintenance DNA methyltransferase (DNMT1) activity. DNMT1 protein level is reduced 50% in CFP1-deficient ES cells. Experiments were performed to investigate the role of CFP1 in regulating maintenance cytosine methylation. Coimmunoprecipitation experiments reveal that endogenous DNMT1 and CFP1 interact in vivo. Protein regions required for the interaction between DNMT1 and CFP1 were mapped. Amino acids 169-493 and 970-1617 of DNMT1 are each sufficient for interaction with CFP1. Three regions spanning the CFP1 protein, amino acids 1-123, 103-367, and 361-656, are each sufficient for interaction with DNMT1. Interestingly, a single-point mutation (C375A) within CFP1 that abolishes the interaction with the Setd1A and Setd1B histone H3K4 methyltransferase complexes does not disrupt the interaction between CFP1 and DNMT1. This result indicates that CFP1 intersects the cytosine methylation machinery independently of its association with the Setd1 complexes.

Selenite reactivates silenced genes by modifying DNA methylation and histones in prostate cancer cells

DNA hypermethylation is a common epigenetic alteration in human prostate cancer and is considered to contribute to development of this disease. Accumulating data suggest that dietary factors may alter cancer risk by modifications of epigenetic processes in the cell. The present study was designed to investigate whether selenium (Se) would alter epigenetic events to regulate methylation-silenced genes in human prostate cancer cells. DNA methylation, histone modifications and gene expression were studied in LNCaP cells after selenite treatment using polymerase chain reaction, western blot analysis, chromatin immunoprecipitation assay and enzymatic activity assay. Our study shows that selenite treatment caused partial promoter DNA demethylation and reexpression of the pi-class glutathione-S-transferase (GSTP1) in LNCaP cells in a dose- and time-dependent manner. Selenite treatment decreased messenger RNA levels of DNA methyltransferases (DNMTs) 1 and 3A and protein levels of DNMT1. Selenite also decreased histone deacetylase activity and increased levels of acetylated lysine 9 on histone H3 (H3-Lys 9), but decreased levels of methylated H3-Lys 9. Selenite treatment reduced levels of DNMT1 and methylated H3-Lys 9 associated with the GSTP1 promoter, but increased levels of acetylated H3-Lys 9 associated with this promoter. Additionally, selenite treatment decreased general DNA methylation and caused partial promoter demethylation and reexpression of the tumor suppressor adenomatous polyposis coli and cellular stress response 1, a gene involving tumor growth and metastasis. Our study demonstrates that Se can epigenetically modulate DNA and histones to activate methylation-silenced genes. These epigenetic modifications may contribute to cancer prevention by Se.

Bortezomib induces DNA hypomethylation and silenced gene transcription by interfering with Sp1/NF-κB–dependent DNA methyltransferase activity in acute myeloid leukemia

Bortezomib reversibly inhibits 26S proteasomal degradation, interferes with NF-kappaB, and exhibits antitumor activity in human malignancies. Zinc finger protein Sp1 transactivates DNMT1 gene in mice and is functionally regulated through protein abundance, posttranslational modifications (ie, ubiquitination), or interaction with other transcription factors (ie, NF-kappaB). We hypothesize that inhibition of proteasomal degradation and Sp1/NF-kappaB-mediated transactivation may impair aberrant DNA methyltransferase activity. We show here that, in addition to inducing accumulation of polyubiquitinated proteins and abolishment of NF-kappaB activities, bortezomib decreases Sp1 protein levels, disrupts the physical interaction of Sp1/NF-kappaB, and prevents binding of the Sp1/NF-kappaB complex to the DNMT1 gene promoter. Abrogation of Sp1/NF-kappaB complex by bortezomib causes transcriptional repression of DNMT1 gene and down-regulation of DNMT1 protein, which in turn induces global DNA hypomethylation in vitro and in vivo and re-expression of epigenetically silenced genes in human cancer cells. The involvement of Sp1/NF-kappaB in DNMT1 regulation is further demonstrated by the observation that Sp1 knockdown using mithramycin A or shRNA decreases DNMT1 protein levels, which instead are increased by Sp1 or NF-kappaB overexpression. Our results unveil the Sp1/NF-kappaB pathway as a modulator of DNA methyltransferase activity in human cancer and identify bortezomib as a novel epigenetic-targeting drug.

Mithramycin A inhibits DNA methyltransferase and metastasis potential of lung cancer cells

Abnormal CpG island hypermethylation of multiple tumor-suppressor genes (TSGs) can lead to the initiation and progression of human cancer. The cytosine of the CpG island on the promoter region is methylated by 5'-cytosine-methyltransferases (DNMTs). Pharmacologic inhibitors of CpG island methylation provide a rational approach to reactivate the TSGs in tumor cells and to restore the critical cellular pathways in cancer cells. Mithramycin A (MMA) is known to be a GC- and CG-rich DNA-binding agent. We sought to determine whether MMA could inhibit CpG island methylation and DNMT expression in lung cancer cells. We found that MMA reduced the CpG island methylation of antimetastasis TSGs, including SLIT2 and TIMP-3 genes, and was associated with the prevention of metastasis. When highly metastatic CL1-5 lung cancer cells were treated with low doses (10 nmol/l) of MMA for 14 days, they reexpressed mRNA levels for these genes. MMA also inhibited the invasion phenotypes of CL1-5 cells as indicated by its inhibition of cancer cell migration using wound-healing and transwell assays. Molecular docking of MMA onto the DNMT1 catalytic domain revealed that MMA might interact with the catalytic pocket of DNMT1. Western blots showed that DNMT1 protein levels were depleted after MMA. These data support the idea that MMA has demethylation and antimetastasis effects on lung cancer cells. This mechanism might be mediated by the interaction of MMA and DNMT1, leading to the depletion of the DNMT1 protein and the reversal of the metastasis phenotype in lung cancer cells.

Phosphatidylinositol 3-kinase/protein kinase B pathway stabilizes DNA methyltransferase I protein and maintains DNA methylation

DNA methylation, which affects gene expression and chromatin stability, is catalyzed by DNA methyltransferases (DNMTs) of which DNMT1 possesses most abundant activity. PI3K/PKB pathway is an important pathway involved in cell proliferation, viability, and metabolism and often disrupted in cancer. Here we investigated the impact of PKB on DNMT1 and DNA methylation. Positive correlation between PKB-Ser473-phosphorylation and DNMT1 protein level in 17 human cell lines (p<0.01) and in 27 human bladder cancer tissues (p<0.05) was found. With activator, inhibitor, siRNA and constitutively active or dominant-negative plasmids of PKB, we found that PKB increased the protein level of DNMT1 without coordinate mRNA change, which was specific rather than due to cell-cycle change. PKB enhanced DNMT1 protein stability independent of de novo synthesis of any protein, which was attributed to down-regulation of N-terminal-120-amino-acids-dependent DNMT1 degradation via ubiquitin–proteasome pathway. Gsk3β inhibitor rescued the decrease of DNMT1 by PKB inhibition, suggesting that Gsk3β mediated the stabilization of DNMT1 by PKB. Then role of PKB regulating DNMT1 was investigated. Inhibition of PKB caused observable DNA hypomethylation and chromatin decondensation and DNMT1 overexpression partially reversed cell growth inhibition by PKB inhibition. In conclusion, our results suggested that PKB enhanced DNMT1 stability and maintained DNA methylation and chromatin structure, which might contribute to cancer cell growth.

29 CXXC-Finger Protein 1 Regulates DNA Methyltransferase 1 Protein Expression and Activity.

Epigenetic regulation refers to changes in gene expression that occur without changes in the nucleotide sequence. Cytosine methylation and histone tail modifications are two epigenetic modifications that influence gene expression. Elucidating epigenetic mechanisms of gene regulation is becoming increasingly important as deregulation of epigenetic processes is observed in many diseases, including cancer. The CXXC1 gene encodes CXXC finger protein 1 (CFP1), a transcriptional activator that specifically binds unmethylated CpG dinucleotides. The specific binding activity of CFP1 makes it unique in that most CpG binding proteins bind methylated CpG dinucleotides and facilitate heterochromatin formation. CFP1 has recently been identified as a member of the mammalian SET1 histone H3 lysine 4 methyltransferase complex. Disruption of CXXC1 in mice results in an early embryonic lethal phenotype, therefore CXXC1−/− embryonic stem (ES) cells were isolated from CXXC1−/− blastocysts to further characterize the function of CFP1. Embryonic stem cells lacking expression of CFP1 exhibit multiple epigenetic modification defects including altered histone modifications and reduced global cytosine methylation. DNA methyltransferase 1 (Dnmt1) is the major source of DNA methyltransferase activity in mammalian cells and is responsible for copying methylation patterns during DNA replication. Dnmt1 protein level and methyltransferase activity are decreased by ~50% in CXXC1−/− ES cells and are rescued by stable expression of murine CFP1. Dnmt1 transcript level is not reduced in CXXC1 −/− ES cells, however. Immunoprecipitation experiments revealed an interaction between CFP1 and Dnmt1 in vivo. Regulation of Dnmt1 protein level and activity by CFP1 is an exciting discovery in that it is the first example of reduced Dnmt1 protein without direct disruption of Dnmt1 gene function. The functional significance of this novel intersection of epigenetic regulatory proteins is the principle focus of ongoing experiments.

Effects of pshRNA-DNMT1 on the proliferation and apoptosis of gastric cancer: experiments in vitro and in vivo

To investigate the effects of pshRNA-DNMT1 on the proliferation and apoptosis of gastric cancer. Recombinant eukaryotic expression plasmid pshRNA-DNMT1 containing the sequence of the gene of DMNT1 that methylates the specific pyrimidine residue in the DNA promoter region was constructed. Human gastric cells of the line AGS were cultured and transfected with pshRNA-DNMT1. Western blotting was used to detect the protein expression of DNMT1 of the AGS cells, and RT-PCR was used to detect the mRNA expression of DNMT1 of the AGS cells. MTT method was used to dynamically monitor the surviving cells and the cell apoptotic was observed by electron microscopy and TUNEL method. Forty nude the mice were inoculated with suspension of AGS cells. When the tumor reached the size of 5 - 6 mm in diameter the mice were randomly divided into 5 equal groups to be injected intravenously with PBS, liposome, pTZU6 + 1, pshRNA-DNMT1 of medium dose, and pshRNA-DNBMT1 of large dose for 4 times with an interval of 3 days. The tumor size was measured every day. Three days after the last injection the mice were killed and the tumors were taken out to undergo light and electron microscopy and TUNEL method to detect the cell apoptosis. Immunohistochemistry was used to detect the proliferating cell nuclear antigen (PCNA) of the cells. The protein and mRNA expression levels of DNMT1 in the cultured AGS cells 24, 48, and 72 hours after transfection of the pshRNA-DNMT1 group were all lower than those of the control group. The numbers of surviving AGS cells of the pshRNA-DNMT1 group became significantly gradually lower than those of the liposome and pTZU6 + 1 groups since 24 hours after transfection (all P < 0.05). The apoptotic rate of AGS cells in the pshRNA-DNMT1 group was 34.78% +/- 0.52%, significantly higher than those of the liposome and pTZU6 + 1 groups (4.86% +/- 0.17% and 5.12% +/- 0.76% respectively, both P < 0.05). The subcutaneous tumors of the mice of the PGS, liposome, and pTZU6 + 1 groups augmented along with time without significant differences among these 3 groups (all P > 0.05). The tumor of the 2 pshDNMT1 groups began to augment since the 5(th) day and began to be reduced in size since the 10(th) day in comparison with the other 3 groups (all P < 0.05), and the tumor size of the pshRNA-DNMT1 (large dose) group was significantly smaller than that of the pshRNA-DNMT1 (medium dose) group 15 days after the injection (P < 0.05). The rates of cell apoptosis of the pshRNA-DNMT1 (large dose) and pshRNA-DNMT1 (medium dose) groups were both significantly higher than those of the other 3 groups (all P < 0.05) and with a sufficient difference between these 2 pshRNA-DNMT1 groups (P < 0.05). PCNA analysis showed that the proliferation activity of the cells in the pshRNA-DNMT1 groups was significantly suppressed. The recombinant plasmid pshRNA-NMT1 effectively and specifically inhibits the expression of the gene DNMT1, thus inhibiting the proliferation and inducing the apoptosis of gastric cancer cells.

A Folate- and Methyl-Deficient Diet Alters the Expression of DNA Methyltransferases and Methyl CpG Binding Proteins Involved in Epigenetic Gene Silencing in Livers of F344 Rats

Aberrations in methylation profile of the genome occur in human cancers induced by folate deficiency. To elucidate the underlying mechanism, male F344 rats were fed a diet deficient in l-methionine and devoid of folic acid and choline (FMD diet), which is known to induce hepatocellular carcinomas. We investigated changes in the DNA methylation machinery, namely, de novo DNA methyltransferases (Dnmt3a and 3b), maintenance DNA methyltransferase (Dnmt1), and methyl CpG binding proteins (MBDs), in rat livers during early stages of tumorigenesis. RT-PCR and Western blot analyses revealed differential expression of these proteins in the livers of rats fed the FMD diet. Although the hepatic Dnmt1 mRNA level declined with age (P < 0.001), it was elevated (P < 0.001) in deficient rats compared with controls. The changes in hepatic Dnmt1 protein level with the diet correlated with its mRNA levels (r = 0.60, P = 0.002). Similarly, the Dnmt3a mRNA level was elevated in rats fed the FMD diet (P < 0.001), whereas the Dnmt3b level (mRNA and protein) was not affected by diet or age. Compared with controls, hepatic MBD1-3 RNA levels increased (P < 0.001) and the protein levels of MBD1, 2, and 4 were elevated (P < 0.001) in the deficient rats. In both diet groups, hepatic MBD2 protein decreased (P < 0.001), whereas MeCP2 protein increased (P < 0.001) with age. These results demonstrate that a combined folate and methyl deficiency alters components of the DNA methylation machinery by both transcriptional and posttranscriptional mechanisms during early stages of hepatocarcinogenesis.

Inhibition of DNA Methyltransferase Activity Prevents Tumorigenesis in a Mouse Model of Prostate Cancer

Transcriptional silencing of tumor suppressor genes by DNA methylation plays an important role in tumorigenesis. These aberrant epigenetic modifications may be mediated in part by elevated DNA methyltransferase levels. DNA methyltransferase 1 (DNMT1), in particular, is overexpressed in many tumor types. Recently, we showed that Dnmt1 is transcriptionally regulated by E2F transcription factors and that retinoblastoma protein (pRb) inactivation induces Dnmt1. Based on these observations, we investigated regulation of Dnmt1 by polyomavirus oncogenes, which potently inhibit the pRb pocket protein family. Infection of primary human prostate epithelial cells with BK polyomavirus dramatically induced Dnmt1 transcription following large T antigen (TAg) translation and E2F activation. For in vivo study of Dnmt1 regulation, we used the transgenic adenocarcinoma of the mouse prostate (TRAMP) model, which expresses the SV40 polyomavirus early region, including TAg, under control of a prostate-specific promoter. Analysis of TRAMP prostate lesions revealed greatly elevated Dnmt1 mRNA and protein levels beginning in prostatic intraepithelial neoplasia and continuing through advanced prostate cancer and metastasis. Interestingly, when TRAMP mice were treated in a chemopreventive manner with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza), 0 of 14 mice developed prostate cancer at 24 weeks of age, whereas 7 of 13 (54%) control-treated mice developed poorly differentiated prostate cancer. Treatment with 5-aza also prevented the development of lymph node metastases and dramatically extended survival compared with control-treated mice. Taken together, these data suggest that Dnmt1 is rapidly activated by pRb pathway inactivation, and that DNA methyltransferase activity is required for malignant transformation and tumorigenesis.

CpG-binding Protein (CXXC Finger Protein 1) Is a Component of the Mammalian Set1 Histone H3-Lys4 Methyltransferase Complex, the Analogue of the Yeast Set1/COMPASS Complex

CpG-binding protein (CXXC finger protein 1 (CFP1)) binds to DNA containing unmethylated CpG motifs and is required for mammalian embryogenesis, normal cytosine methylation, and cellular differentiation. Studies were performed to identify proteins that interact with CFP1 to gain insight into the molecular function of this protein. Immunoprecipitation and mass spectrometry reveal that human CFP1 associates with a approximately 450-kDa complex that contains the mammalian homologues of six of the seven components of the Set1/COMPASS complex, the sole histone H3-Lys4 methyltransferase in yeast. In vitro assays demonstrate that the human Set1/CFP1 complex is a histone methyltransferase that produces mono-, di-, and trimethylated histone H3 at Lys4. Confocal microscopy reveals that CFP1 and Set1 co-localize to nuclear speckles associated with euchromatin. A Set1 complex of reduced mass persists in murine embryonic stem cells lacking CFP1. These cells carry elevated levels of methylated histone H3-Lys4 and reduced levels of methylated histone H3-Lys9. Together with the previous finding of reduced levels of cytosine methylation, these data indicate that cells lacking CFP1 contain reduced levels of heterochromatin. Furthermore, ES cells lacking CFP1 exhibit a 4-fold excess of histone H3-Lys4 methylation following induction of differentiation, indicating that CFP1 restricts the activity of the Set1 histone methyltransferase complex. These results reveal a mammalian counterpart to the yeast Set1/COMPASS complex. The presence of CFP1 in this complex implicates this protein as a critical epigenetic regulator of histone modification in addition to cytosine methylation and reveals one mechanism by which this protein intersects with the epigenetic machinery.

Phase I Study of Low Dose Decitabine in Patients with Acute Myeloid Leukemia (AML): Pharmacokinetics (PK), Pharmacodynamics (PD), and Clinical Activity.

Decitabine, an inhibitor of DNA methyltransferase (DNMT) enzymes, has clinical activity in myeloid malignancies, even at low doses (>1 log below the MTD) which may be optimal for demethylation rather than only cytotoxicity. We designed a two step trial for AML patients (pts) to determine: Step 1) the minimal effective pharmacologic dose (MEPD) of decitabine as a single agent (defined as lowest dose to induce reexpression of genes commonly methylated in AML in 5/6 pts treated at a single dose level) and Step 2) the MTD of the histone deacetylase inhibitor valproic acid given in combination with decitabine at this MEPD. To date, Step 1 is ongoing; decitabine alone has been administered to 14 pts at two dose levels. Pts had relapsed/refractory AML (N=8) or age>60 and ineligible/refused standard induction therapy (N=6). Pts ranged in age from 57–83 years and had received ≥ 2 prior inductions (N=6), 1 prior induction (2), or were untreated (4). 8 pts received decitabine at 15mg/m2/IV over 1 hr daily (d) for 10 consecutive d, and 6 received 20mg/m2/d on the same schedule, every 28 d. Plasma decitabine levels were analyzed by a validated LC-MS/MS method with a sensitivity of 2 ng/ml developed in our laboratory. Plasma drug concentration-time courses on d 1 and 10 achieved a mean Cmax of 93 ng/ml (N=7) and followed a two compartment infusion model. The mean short and long half-lives were 2.7 and 36.9 min, respectively, with a trend of decreasing the longer half-life on d 10. According to published bone marrow (BM) response criteria (Cheson, JCO 2003), 4/10 pts who completed two cycles of therapy responded (2 at each dose level). 3 had complete response with incomplete recovery of counts (CRi), and one achieved CR. Hematologic improvement (HI) or clinical benefit was seen in 3 additional pts: one achieved neutrophils of 2,200/uL, platelets (plts)>100,000/uL, disappearance of circulating blasts, and disappearance of BM blasts except by flow cytometry; one pt achieved plt transfusion independence (plts>100,000/uL); one pt had stabilization of disease for 6 months. Two more pts who remain on study are not yet evaluable for BM response but have already had significant HI with plts of 259,000 and 74,000/uL, respectively, after one cycle of therapy. Two pts were not evaluable for response due to death from sepsis or death related to decitabine-induced differentiation syndrome (first reported by Blum, ASH 2004), respectively. Decitabine was well tolerated, given typically as an outpatient. There were no severe non-hematologic drug related toxicities. Pts with HI or BM response required plt transfusion support when subsequent cycles were administered. At 15mg/m2/d x 10 d: 4/6 pts experienced at least 100% increase in reexpression genes commonly methylated in AML such as p15 or estrogen receptor; immunoblotting demonstrated DNMT1 protein depletion in 4/6 pts lasting 4–42 d. Clinical trial design based on achieving the MEPD is feasible in AML; we demonstrate clinical activity of low dose decitabine associated with changes in levels of the DNMT1 target and reexpression of epigenetically silenced genes. Complete PK and PD studies including gene reexpression by quantitative RT-PCR, DNMT1 protein levels by immunoblotting, and decitabine-induced promoter demethylation by COBRA/bisulfite sequencing will be presented. (NCI U01 CA 76576-05)

Suppression of DNA methyltransferase 1 levels in head and neck squamous carcinoma cells using small interfering RNA results in growth inhibition and increase in Cdk inhibitor p21

The ectopic expression of DNA methyltransferase 1 (DNMT 1) can transforms mammalian cells, and the inhibition of DNMT1 activity reverses that phenotypic transformation. Therefore, DNMT1 is considered to be an excellent target for therapeutic intervention. Previously, inhibition of DNMT1 was accomplished by using an antagonist or by antisense oligonucleotides. In this study, we examined the ability of the novel approach using small interfering RNA (siRNA) to disrupt the expression of DNMT1 in human non-small cell lung carcinoma A549 cells and the consequences of such an intervention. Transfection of DNMT1 siRNA decreased DNMT1 protein levels specifically and effectively. This decrease was accompanied by suppression of cell proliferation and colony-forming ability. The mechanism of this inhibition may be related to the increased levels of the cyclin dependent kinase inhibitor p21. These results suggest that the siRNA approach can be used to disrupt effectively DNMT1 activity and cancer cell growth.