Findings in epigenetic changes in meylodysplastic syndromes (MDS) and the development of demethylating drugs provide a new approach to the treatment of MDS. We used standard-dose decitabine for treatment of MDS in an elderly patient with an International Prostate Symptom Score (IPSS) of moderate risk group 2, and achieved a complete response in the first course. We report our experience with this case and review the relevant literatures.
Azacitidine ; analogs & derivatives ; therapeutic use ; DNA Modification Methylases ; antagonists & inhibitors ; Female ; Humans ; Middle Aged ; Myelodysplastic Syndromes ; drug therapy

OBJECTIVETo compare the sensitivity of different hepatocellular carcinoma (HCC) cell lines (HepG2, QGY7701, HepG2.2.15) and the normal liver cell line L02 to 5-aza-dC, an DNA methyltransferase inhibitor, and to explore the relationship between global DNA methylation level and the sensitivity to 5-aza-dC.

METHODSHepG2, QGY7701, HepG2.2.15 and L02 cells were treated with 5-aza-dC at different concentration, cell proliferation was measured by MTT method, cell apoptosis was detected by measuring caspase 3 activity and cellular DNA fragmentation ELISA.

RESULTSCompared to HepG2 and QGY7701 cells, HepG2.2.15 were less sensitive to the treatment of 5-aza-dC; the normal liver cell line L02 was less sensitive to 5-aza-dC than the HCC cell lines.

CONCLUSIONSThe sensitivity to 5-aza-dC of HCC cell lines and normal liver cells is not correlated with the global DNA methylation level.

Azacitidine ; analogs & derivatives ; pharmacology ; Carcinoma, Hepatocellular ; metabolism ; Caspase 3 ; metabolism ; DNA Methylation ; drug effects ; DNA Modification Methylases ; antagonists & inhibitors ; Hep G2 Cells ; Humans

Although the traditional chemotherapy has achieved a certain effect for patients with acute myeloid leukemia (AML), but there are still limitations in terms of improving the rate of complete remission and overcome relapse after remission. The further study found that many cytogenetic molecular and epigenetic abnormalities occurred during the progression of AML, such as abnormal expression of cell surface molecules, mutation, gene aberrant methylation and so on. The drugs targeted at these changes can improve the prognosis for patients, and provide a new way for treating patients with AML. At present, the mostly targeted drugs include monoclonal antibodies CD33-Ab, tyrosine kinase inhibitor, inhibitors of DNA methyltransferases inhibitors and so on. In this review, the progress of targeted therapy in AML treatment is summarized.
Antibodies, Monoclonal ; therapeutic use ; DNA Modification Methylases ; antagonists & inhibitors ; Humans ; Leukemia, Myeloid, Acute ; drug therapy ; Mutation ; Prognosis ; Protein Kinase Inhibitors ; therapeutic use ; Remission Induction

To study the methylation in the promoter of LRP15 gene and its relationship with gene expression and to explore the possible mechanism of regulating LRP15 gene methylation, the methylation in the promoter of LRP15 gene in K562 cell line was detected by MS-PCR. Then K562 was exposed to 5-aza-2'-deoxycytidine (CdR) and trichostatin (TSA), to determine whether the silencing of LRP15 gene by de novo methylation could be reversed. As a result, it was confirmed by MS-PCR that the promoter of LRP15 was hypermathylated in K562 cell line, and lost its transcription activity. After CdR, with or without TSA, the silencing of LRP15 gene by de novo methylation can be reversed. Observation demonstrated that the expression of LRP15 was controlled by methylation in its promoter in K562. It is suggested that methyltransferase inhibitor and deacetylase inhibitor may be effective agents in leukemia therapy.
Azacitidine ; analogs & derivatives ; pharmacology ; DNA Methylation ; DNA Modification Methylases ; antagonists & inhibitors ; Enzyme Inhibitors ; pharmacology ; Gene Expression Regulation ; drug effects ; Histone Deacetylase Inhibitors ; Humans ; Hydroxamic Acids ; pharmacology ; K562 Cells ; Neoplasm Proteins ; biosynthesis ; genetics ; Polymerase Chain Reaction ; methods ; Promoter Regions, Genetic ; genetics

DNA methyltransferase inhibitor, 5-azacitidine (AC) is effective in myelodysplastic syndromes (MDS) and can induce re-expression in cancer. We analyzed the methylation of 25 tumor suppressor genes in AC-treated MDS. Hypermethylation of CDKN2B, FHIT, ESR1, and IGSF4 gene was detected in 9/44 patients. In concordance with the clinical response, a lack of or decreased methylation in 4 patients with hematologic improvements and persistent methylation in 4 others with no response was observed. The mRNA expression of CDKN2B, IGSF4, and ESR1 was significantly reduced in MDS. Our results suggest that methylation changes contribute to disease pathogenesis and may serve as marker to monitor the efficacy of treatments.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Azacitidine/*pharmacology/*therapeutic use ; DNA Methylation/*drug effects ; DNA Modification Methylases/antagonists & inhibitors/metabolism ; Enzyme Inhibitors/*pharmacology/*therapeutic use ; Female ; Genes, Tumor Suppressor ; Humans ; Male ; Middle Aged ; Myelodysplastic Syndromes/*drug therapy/*genetics ; Young Adult

OBJECTIVETo study the regulation of methylation inhibitor 5-aza-2'-deoxycytidine on transcription of EphB4 gene and effects on the proliferation and apoptosis of human acute lymphocyte leukemia cell line CEM.

METHODSBisulfite sequencing PCR was used to detect CpG island methylation density in EphB4 promoter. The expression of EphB4 mRNA and protein was determined by Q-PCR and Western blot. MTS assay and flow cytometry were used to detect the apoptosis of CEM cells after treatment with different concentrations of 5-aza-2'-deoxycytidine (1.0, 2.5 and 5 μmol/L).

RESULTSMethylation of EphB4 gene promoter was detected in CEM cells (31.4%). The methylation level of EphB4 gene was down-regulated after treatment with various concentrations of 5-aza-2'-deoxycytidine. The EphB4 mRNA and protein expression in CEM cells increased after 5-aza-2'-deoxycytidine treatment. 5-Aza-2'-deoxycytidine significantly inhibited the cell growth in dose and time dependent manners. Early apoptosis rates of CEM cells increased from 4.1% to 24.8% 96 hrs after 5-aza-2'-deoxycytidine treatment. CEM cells in G1 phase decreased from 62.4% to 46.8%, cells in G2 phase increased from 2.1% to 16.2%, and CEM cells were arrested in G2 phase after treatment with 5 μmol/L 5-aza-2'-deoxycytidine for 96 hrs.

CONCLUSIONS5-Aza-2'-deoxycytidine, an inhibitor of specific methylation transferase, can induce expression of the silent EphB4 gene in CEM cells, inhibit the proliferation of leukemia cells and induce cell apoptosis.

Apoptosis ; drug effects ; Azacitidine ; analogs & derivatives ; pharmacology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; DNA Methylation ; DNA Modification Methylases ; antagonists & inhibitors ; Humans ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; drug therapy ; pathology ; RNA, Messenger ; analysis ; Receptor, EphB4 ; genetics

BACKGROUNDGene therapy and epigenetic therapy have gained more attention in cancer treatment. However, the effect of a combined treatment of gene therapy and epigenetic therapy on head and neck squamous cell carcinoma have not been studied yet. To study the mechanism and clinical application, human laryngeal carcinoma cell (Hep-2) tumor-bearing mice were used.

METHODSA xenograft tumor model was established by the subcutaneous inoculation of Hep-2 cells in the right armpit of BALB/c nu/nu mice. The mice with well-formed tumor were randomly divided into six groups. Multisite injections of rAd-p53 and/or 5-aza-dC were used to treat tumor. Tumor growth was monitored by measuring tumor volume and growth rate. p53 and E-cadherin protein levels in tumor tissues were detected by immunohistochemical staining. The mRNA levels were monitored with FQ-PCR.

RESULTSGene therapy was much more effective than single epigenetic therapy and combined therapy. The gene therapy group has the lowest tumor growth rate and the highest expression levels of p53 and E-cadherin.

CONCLUSIONSThe combined treatment of gene and epigenetic therapy is not suggested for treating head and neck carcinoma, because gene therapy shows an antagonistic effect to epigenetic therapy. However, the mechanisms of action are still unclear.

Animals ; Azacitidine ; analogs & derivatives ; therapeutic use ; Cadherins ; analysis ; DNA Modification Methylases ; antagonists & inhibitors ; Epigenesis, Genetic ; Genes, p53 ; Genetic Therapy ; Humans ; Laryngeal Neoplasms ; genetics ; pathology ; therapy ; Male ; Mice ; Mice, Inbred BALB C ; Tumor Suppressor Protein p53 ; analysis ; Xenograft Model Antitumor Assays

OBJECTIVE: To analyze the effect of DNA hypermethylation on NOR1 promoter activity and expression. METHODS: NOR1 promoter plasmids were treated with SssI methyltransferase. The plasmids were modified by sodium bisulfite and purified. Sodium bisulfite-modified plasmids were subjected to PCR with primers designed to analyze the methylation status of 26 CpG sites in a 311-bp region of the NOR1 promoter. Cells were transfected by methylated or mock-methylated promoter plasmids. The promoter activities were assessed by the luciferase levels of cell lysates or by directly observing GFP expression under fluorescence microscope. HL60 cells were treated with different concentrations of 5-aza-dC. Total RNA was isolated from harvested cells. Real-time RT-PCR was used to measure the expression level of NOR1 mRNA. RESULTS: Bisulfite sequencing confirmed that SssI methyltransferase treatment successfully resulted in intensive hypermethylation of the NOR1 promoter plasmids. The promoter activity of NOR1 promoter plasmids was totally blocked by SssI methyltransferase treatment. NOR1 expression levels in HL60 cells were restored by 5-aza-dC treatment. CONCLUSION NOR1 promoter plasmids are intensively hypermethylated by SssI methyltransferase treatment. The promoter activity of NOR1 promoter plasmids are totally blocked by SssI methyltransferase treatment. The 5-aza-dC treatment may restore the endogenous NOR1 mRNA level in HL60 cells.
Azacitidine ; analogs & derivatives ; pharmacology ; Base Sequence ; Cell Line, Tumor ; CpG Islands ; DNA Methylation ; DNA Modification Methylases ; antagonists & inhibitors ; DNA-Cytosine Methylases ; pharmacology ; Decitabine ; Epigenesis, Genetic ; Gene Silencing ; HL-60 Cells ; Humans ; Membrane Transport Proteins ; genetics ; metabolism ; Molecular Sequence Data ; Nasopharyngeal Neoplasms ; pathology ; Promoter Regions, Genetic ; genetics ; RNA, Messenger ; genetics ; metabolism

OBJECTIVETo investigate the change in the expression of E-Cadherin of human hepatocarcinoma cell line SMMC-7721 after transfection by antisense human DNA MTase gene.

METHODSDNA MTase gene eukaryotic expression vectors, including sense and antisense fragments, were constructed with recombinant technology and transfected into the hepatocarcinoma cell line SMMC-7721 with liposome DOTAP. The expression of DNA MTase gene mRNA and E-Cadherin gene mRNA was examined with RT-PCR and the expression of E-Cadherin with immunohistochemical and flow cytometry. The status of methylation in E-Cadherin gene promoter area was examined with methylation specific PCR (MSP).

RESULTSThe sense and antisense eukaryotic expression vectors were successfully constructed and then the constructed recombinant plasmids were successfully transfected into SMMC-7721 cell with liposome DOTAP. The expression of endogenous DNA MTase mRNA was obviously decreased with E-Cadherin gene mRNA and its activity increased in the SMMC-7721 cell, which was tranfected with antisense DNA MTase gene fragment. Moreover, demethylation in the promoter area of E-Cadherin gene was observed with MSP.

CONCLUSIONDemethylation in the promoter area and increasing mRNA level of E-Cadherin gene can be induced by expression inhibition of DNA MTase gene of SMMC-7721 cell line.

Cadherins ; genetics ; metabolism ; Carcinoma, Hepatocellular ; pathology ; CpG Islands ; DNA ; drug effects ; metabolism ; DNA Methylation ; DNA Modification Methylases ; antagonists & inhibitors ; DNA, Antisense ; genetics ; pharmacology ; Gene Expression ; drug effects ; Humans ; Liver Neoplasms ; pathology ; Promoter Regions, Genetic ; drug effects ; RNA, Messenger ; drug effects ; metabolism ; Transfection ; Tumor Cells, Cultured

OBJECTIVETo assess the role of methylated mismatch repair (MMR) genes (hMLH1, hMSH2 and hMSH3) in the carcinogenesis and progression of hepatocellular carcinoma (HCC).

METHODSSamples of 38 cases of HCC along with their corresponding noncancerous tissues, 2 samples of donated normal tissue and 6 cell lines were collected and subject to the methylation-specific PCR (MSP) to examine promoter methylation status of MLH1, MSH2 and MSH3. Six tumor cell lines were analyzed before and after 5-aza-2'-deoxycytidine treatment. In addition, alterations of mRNA expression of MMRs were investigated by quantitative reverse transcription-PCR.

RESULTSCpG island methylation of hMLH1 and hMSH2 was observed in 13.2% (5 of 38 samples) and 68.4% (26 of 38 samples) respectively in HCC, 2.6% (1 of 38 samples) and 55.3% (21 of 38) respectively in corresponding noncancerous tissues, but not in normal control tissues. Promoter methylation of the hMSH2 gene was present in 83.3% of cell lines tested (5/6), but none were observed for the hMLH1 gene. Promoter methylation of the hMSH3 gene was not identified in any tissue samples or cell lines. After 5-aza-2'-deoxycytidine treatment, hMSH2 methylation was induced or completely reversed, and its mRNA expression was increased in most cell lines.

CONCLUSIONSOur results suggest that promoter hypermethylation of hMLH1 and hMSH2 genes is common in HCC. Particularly, there is a high frequency of methylation of hMSH2 in both cancer and noncancerous tissues, but not in normal control tissue. Therefore, hypermethylation of MMR genes, especially hMSH2, may be involved in the carcinogenesis of HCC and may serve as an early diagnostic marker for HCC. The close correlation between hMSH2 methylation and low expression of its mRNA suggests that hMSH2 methylation is an important pathway in the regulation of gene expression.

Adaptor Proteins, Signal Transducing ; Azacitidine ; analogs & derivatives ; pharmacology ; Base Pair Mismatch ; genetics ; Carcinoma, Hepatocellular ; genetics ; Carrier Proteins ; biosynthesis ; genetics ; Cell Line, Tumor ; DNA Methylation ; DNA Modification Methylases ; antagonists & inhibitors ; DNA Repair ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Liver Neoplasms ; genetics ; MutL Protein Homolog 1 ; MutL Proteins ; Neoplasm Proteins ; biosynthesis ; genetics ; Nuclear Proteins ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics