Chromium ; toxicity ; DNA Methylation ; drug effects ; Epigenesis, Genetic ; Humans

Cell Proliferation ; drug effects ; DNA Methylation ; drug effects ; Humans ; K562 Cells ; Stilbenes ; pharmacology ; WT1 Proteins ; metabolism

OBJECTIVETo review the emerging role of DNA methylation inhibitors in cancer therapy and make a serious reflection on their current status and future perspectives.

DATA SOURCESThe data used in the present article were mainly from PubMed with relevant English papers published from April 1988 to January 2010. The search terms were "DNA methylation", "demethylation" and "cancer".

STUDY SELECTIONStudies involved in the DNA methylation in carcinogenesis and DNA methylation inhibitors for cancer therapy were selected. The original milestone articles were also included.

RESULTSTreatment with DNA methylation inhibitors leads to demethylation of a panel of tumour suppressor genes and reverse the expression in different tumors, thus making them potential cancer therapeutics. However, we cannot be very optimistic about their future perspectives because we still have a long way to go before they function well like specific targeted anticancer drugs as we expected.

CONCLUSIONThe best way forward is to further clarify the exact methylation profiles of tumors and to develop novel agents targeting the specific genes.

Antineoplastic Agents ; pharmacology ; therapeutic use ; DNA Methylation ; drug effects ; Humans ; Neoplasms ; drug therapy ; genetics

OBJECTIVETo investigate the effects of neonatal exposure of DNA methylation inhibitor, Cadmium and PCB153 on DNA methylation, apoptosis and spermatogenesis in SD rats.

METHODSNeonatal SD rats were randomly divided into 10 groups and received oral administrations of PCB153 (0.025, 0. 250, 2.500 mg/kg), or Cadmium (1, 2, 4 mg/kg), or positive control 5-Aza-CdR (0.025, 0.250 mg/kg), or vehicle control for five days from PND3. Half of the rats were killed 24 h after the last administration. The remains were fed until 12 weeks. Sperm numbers, apoptosis and DNA methylation levels in testis were investigated.

RESULTSThe daily sperm production was significantly decreased in each neonatal exposed group (P < 0.05). Neonatal rats exposed to 5-Aza-CdR and Cadmium reduced the global DNA methylation level, increased apoptosis, while PCB153 exposure did not significantly change DNA methylation and apoptosis.

CONCLUSIONNeonatal rats exposed to chemicals could reduce spermatogenesis via multiple pathways. Lower DNA methylation and increased neonatal apoptosis were suggested as one of the causes.

Animals ; Animals, Newborn ; Apoptosis ; drug effects ; Cadmium ; toxicity ; DNA Methylation ; drug effects ; Male ; Polychlorinated Biphenyls ; toxicity ; Rats ; Rats, Sprague-Dawley ; Spermatogenesis ; drug effects ; Testis ; drug effects ; metabolism ; pathology

OBJECTIVETo study the antitumour effects of sodium valproate (VPA) on the proliferation, differentiation and cell cycle of Molt-4 cell and to investigate its demethylation mechanisms.

METHODSAfter Molt-4 cells trated with VPA at different concentrations, cell viability and growth curve were assessed by MTT assay. Cell cycle changes were analyzed by flow cytometry. The expression level of p15, DNA methyltransferase 1 (DNMT-1), DNMT3A and 3B mRNA were detected by RT-PCR and the methylation level was detected by hn-MSPCR.

RESULTSVPA significantly inhibited the proliferation of Molt-4 cells. After 48 h culture with 5.0 mmol/L VPA, the percentages of Molt-4 cells in G(0)/G(1) phase was (66.87 ± 3.31)% and in S phase was (8.47 ± 2.56)%, while in control group, the cells in G(0)/G(1) phase increased and in S phase decreased significantly. The p15 gene in Molt-4 cells failed to express due to its hypermethylation. The expression level of p15 gene mRNA increased significantly after exposure to VPA for 48 h. As compared with control group, the expression of DNMT-1 was down-regulated in a dose-dependent manner. The expression level of DNMT3B decreased at 10.0 mmol/L concentration.

CONCLUSIONVPA has a demethylation effect on p15 INK4B gene by inhibiting the DNMT-1 and DNMT3B gene activities to recover p15 gene activity, which arrests Molt-4 cell in G(0)/G(1) phase.

Cell Cycle ; drug effects ; Cell Line, Tumor ; DNA Methylation ; drug effects ; RNA, Messenger ; genetics ; Valproic Acid ; pharmacology

OBJECTIVETo investigate the effects of PHI on histone acetylation and methylation in hepatocellular carcinoma line SMMC-7721 cells.

METHODSApoptosis was measured by TUNNEL assay. Histone methylation and acetylation were detected by Western blot.

RESULTSPHI inhibited cells growth and induced apoptosis. PHI treatment resulted in increased acetylation of histone H3 and H4 , elevated level of histone H3 lysine 4 methylation, and decreased level of histone H3 lysine 9 methylation.

CONCLUSIONSPHI can modulate both histone acetylation and methylation, which could remodel chromatin structure. PHI may be a novel anticancer drug.

Acetylation ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Histones ; metabolism ; Humans ; Isothiocyanates ; pharmacology ; Methylation

OBJECTIVETo study methylation of Id4 gene and demethylation effect of arsenic trioxide (ATO) in Raji cells.

METHODSHuman Burkitt's Raji lymphoma cells were cultared and treated with ATO at different concentrations and different time points. Methylated degree of Id4 gene was detected by methylation specificity polymerase chain reaction (MS-PCR), Id4 mRNA expression in Raji cell by reverse transcription polymerase chain reaction (RT-PCR), the growth of cell by MTT assay, and cell apoptosis and cycle distribution by Flow Cytometry (FCM).

RESULTS(1) The Id4 gene exhaustive methylation in control group, and hypermethylation in experimental group were reversed by ATO in a dose-dependent manner. (2) Id4 mRNA expression in Raji cells treated with ATO for 48 h increased gradually with ATO concentration increasing in experimental group. (3) Raji cell growth inhibited rates after different concentrations of ATO treatment for 24, 48, 72 h were 12.15% ∼ 92.17% in the experimental group (P < 0.05). (4) Apoptosis peak emerged after ATO treatment for 48 hours in experimental group, while a much lower apoptosis in control group. (5) After ATO treatment for 48 h in experiment group, the cells were arrested at G(0)/G(1) phase in a dose-dependent manner.

CONCLUSIONId4 gene presents exhaustive methylation in Raji cells. ATO can reverse the hypermethylation of Id4 gene and recover the expression of Id4 mRNA. Hypermethylation of Id4 gene is one of the reasons of Raji cells malignant proliferations.

Apoptosis ; drug effects ; Burkitt Lymphoma ; genetics ; Cell Line, Tumor ; DNA Methylation ; Humans ; RNA, Messenger ; genetics

OBJECTIVETo detect the expression changes of the demethylase TETs (Ten-eleven translocation enzymes) in human embryonic kidney cell (HEK293) exposed to high dose cadmium chloride (CdCl2), and to investigate the regulation effects of TETs on global genomic methylation.

METHODSHEK293 cells were exposed to CdCl2 for 24 h, 48 h and 72 h, the survival rate was tested by CCK-8 (cell counting kit-8) method, and the cell morphology was observed. The levels of TETs mRNA and protein were detected by fluorescence quantitative PCR and Western blot, respectively. The genomic DNA methylation level was detectedby pyro sequencing assay.

RESULTSCdCl2 had toxic effects on HEK293 cells, and the half inhibitory concentration (IC50) was 1.78 µmol/L. After exposure of CdCl2 for 24 h, 48 h and 72 h, the morphology of HEK293 cells was altered, and the high dose group (2.0 µmol/L) showed vacuolar changes and fuzzy appearance. The level of TET1 mRNA in groups of 0.0, 0.5, 1.0, 2.0 µmol/L were 0.23 ± 0.13, 0.48 ± 0.12, 0.59 ± 0.16 and 0.95 ± 0.39, respectively (F = 182.89, P = 0.002); The level of TET2 mRNA in groups of 0.0, 0.5, 1.0, 2.0 µmol/L were 0.23 ± 0.12, 0.32 ± 0.02,0.31 ± 0.10 and 0.34 ± 0.07, respectively (F = 27.94, P < 0.001); The level of TET3 mRNA in groups of 0.0, 0.5, 1.0, 2.0 µmol/L were 0.26 ± 0.10, 0.27 ± 0.11, 0.25 ± 0.11 and 0.28 ± 0.09, respectively (F = 1.76, P = 0.036). The interaction effect existed between exposure time and doses of TET1 mRNA, TET2 mRNA and TET3 mRNA (F values were 32.94, 23.04 and 13.78, respectively; P values were < 0.001, 0.041 and < 0.001, respectively). Western blot showed that in different exposure time and dose, the protein expression levels of TETs had the similar trend as mRNA levels. In 24 h (55.01 ± 3.62)%, 48 h (48.31 ± 8.99)%, 72 h (48.76 ± 6.60)%, the DNA methylation had significant differences (F = 18.50, P < 0.001); In groups of 0.0 µmol/L (55.29 ± 2.83)%, 0.5 µmol/L (55.35 ± 3.11)%, 1.0 µmol/L (48.58 ± 6.40)% and 2.0 µmol/L (43.56 ± 7.89)%, the differences of DNA methylation had significant differences (F = 7.03, P = 0.048); the effect of interaction was also existed (F = 2.73, P = 0.043).

CONCLUSIONIn the short term exposure to CdCl2, the levels of TETs mRNA and protein showed a trend of increase according to the exposure time and dose, and the methylation level of whole genomic DNA was also altered. The demethylase TETs may play a role in regulating the genomic methylation level of HEK293 exposed to cadmium.

Cadmium Chloride ; toxicity ; DNA Methylation ; Dioxygenases ; genetics ; Epithelial Cells ; drug effects ; HEK293 Cells ; Humans ; RNA, Messenger

This study was aimed to investigate the effect of 5-Aza-CdR on the biological activity of human erythroleukemia cell line K562 and the expression of inhibitor of DNA binding 4 (ID4). ID4 methylation in K562 cell line was detected by methylation-specific PCR. RQ-PCR was used to analyze the expression levels of ID4 mRNA in K562 cell line treated by different concentrations of 5-Aza-CdR. Cell apoptosis rate and cell cycle were analyzed by flow cytometry. The result showed that ID4 gene methylation existed in K562 cells, ID4 mRNA expression in K562 cells treated with 5-Aza-CdR increased in a concentration-dependent manner, the difference between experimental groups was statistical significant (p < 0.01). The 5-Aza-CdR could enhance the apoptotic rate of K562 cells in time and dose-dependent manner, the apoptotic rate of K562 cells highly correlated to relative expression level of ID4 mRNA (r = 0.95). After the K562 cells were treated by 5-Aza-CdR for 48 hours, cells in G(0)/G(1) phase increased, cells in G(2)/M phase decreased along with enhancement of drug concentration. It is concluded that methyltransferase inhibitor 5-Aza-CdR can re-express the silent ID4 gene in K562 cells. The upregulation of ID4 may be a key factor to give rise to cell apoptosis, and the cell cycle of K562 cells can be arrested by 5-Aza-CdR.
Apoptosis ; drug effects ; Azacitidine ; analogs & derivatives ; pharmacology ; Cell Cycle ; Cell Proliferation ; drug effects ; DNA Methylation ; Gene Expression ; drug effects ; Humans ; Inhibitor of Differentiation Proteins ; genetics ; K562 Cells

The aim of this study was to investigate the effects of H3K27 methylation inhibitor EPZ005687 on the apoptosis, proliferation and cell cycle of U937 cells and normal CD34⁺ cells. The U937 cells and normal CD34⁺ cells were treated with different concentration of EPZ005687 at different time points. The apoptosis rate was determined by Annexin V/PI staining. The cell proliferation and cell cycle was determined using WST-1 assay and 7-AAD assay, respectively. The activity of H3K27 methylation was detected by chemiluminescent immunoassay. The results showed that the EPZ005687 induced an obvious apoptosis of U937 cells. The apoptotic rate was 3.96% ± 0.79%,5.74% ± 0.73%,13.34% ± 1.77% and 25.24% ± 2.55% in U937 cells treated with 0.5, 1, 5 and 10 µmol/L EPZ005687 for 48 hours, respectively. However, EPZ005687 had rare effect on normal bone marrow(NBM) CD34⁺ cells. The apoptotic rate was 3.64% ± 0.62%,4.28% ± 0.99%,6.18% ± 1.19% and 7.56% ± 1.34% after U937 cells were treated with 0.5, 1, 5 and 10 µmol/L EPZ005687 for 48 hours, respectively. EPZ005687 inhibited obviously the proliferation of U937 cells but had weak effect on the proliferation of NBMCD34⁺ cells. The inhibitory effect of EPZ005687 on U937 cells was time-dependent after treated with 0.5, 1, 5 and 10 µmol/L EPZ005687 from 12 to 96 hours. EPZ005687 induced G1 phase blocking (G1%, 64.18% ± 13.27% vs 49.43% ± 12.54%) and decreased the percentage of cells in S phase (9.67% ± 2.61% vs15.26% ± 5.58%) in U937 cells. However, EPZ005687 had no effect on the cell cycle of NBMCD34⁺ cells. In addition, EPZ005687 produced obviously depletion of H3K27 methylation in U937 cells (P < 0.05), but hardly had effect on the H3K27 methylation of NBMCD34⁺ cells. It is concluded that the EPZ005687 inhibites proliferation, induces apoptosis and cell cycle blocking in G1 phase in leukemia cells. This agent may have potential value in clinical application.
Antigens, CD34 ; metabolism ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Humans ; Indazoles ; pharmacology ; Methylation ; Pyridones ; pharmacology ; U937 Cells