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 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

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 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 investigate the effect of the exposure to di- (2-ethylhexyl) phthalate (DEHP) during pregnancy on the DNA methylation level of genomes in the testis of the offspring in mice.

METHODSPregnant KM mice were randomly divided into three groups, normal control, corn oil and DEHP-exposed. Corn oil and DEHP (500 mg/[kg x d]) were administrated respectively from gestation day 12.5 (GD 12.5) to postnatal day 3 (PND 3). The testes of the offspring were excised on PND 7, and their genomic DNA was treated with EcoR I /Msp I and EcoR I /Hpa II. The genome-wide DNA methylation patterns of the CCGG sites were detected by methylation-sensitive amplification polymorphism (MSAP). The samples were electrophoresed in the ABI 3730 DNA sequencer and the results analyzed by the Genescan3.1.

RESULTSThe average incidence of DNA methylation was (34.03 +/- 3.05)% in the DEHP-exposed mice, obviously higher than (28.37 +/- 2.37)% in the normal control and (28.58 2.45)% in the corn oil group, with statistically significant differences (P < 0.05).

CONCLUSIONExposure to DEHP during pregnancy increases the DNA methylation level of the genome in the testis of the offspring and affects the apparent genetic modification of the genome, which may be one of the important toxicological causes of the lesion in the reproductive system.

Animals ; DNA Methylation ; drug effects ; Diethylhexyl Phthalate ; pharmacology ; Female ; Genome ; Male ; Mice ; Mice, Inbred Strains ; Pregnancy ; Prenatal Exposure Delayed Effects ; Random Amplified Polymorphic DNA Technique ; Testis ; drug effects

More and more studies have found that the occurrence of tumors are directly related to the abnormal expression of oncogene and antioncogene. If the antioncogene is mutated or absent, the function of cells will be weakened and inactivated, the cells will be duplicated repeatedly out of control, then will induce occurrence and metastasis of tumor. For example, SHP-1 tyrosine phosphatase, as an antioncogene, is a key negative regulator in signaling transduction of haematopoietic cells. The decrease and silence of SHP-1 play an important role in tumorigenesis. If the oncogene in leukemia patients lost the effect of negative regulation of antioncogene, the oncogene would be expressed abnormally high, such as the oncogene c-kit (an important member of the class III in the tyrosine kinase receptor family) in many kinds of leukemia cells expresses actively. Studies have shown that the high methylation of promoter region would induce the inactivation of tumor suppressor and active expression of oncogene, therefore, the restoring normal methylation of promoter region will contribute to restoration of normal gene expression, thus achieving the purpose of gene therapy for leukemia. In this article, the methylation, methylation abnormality and leukemia, methylation suppressors and therapy of leukemia are briefly reviewed.
DNA Methylation ; drug effects ; Humans ; Leukemia ; drug therapy ; metabolism ; Protein Tyrosine Phosphatase, Non-Receptor Type 6 ; metabolism

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