OBJECTIVE: To analyze the relationship between and gene methylation with aging in the general population. METHODS: We collected peripheral blood samples from 284 male and 246 female healthy subjects for detection of methylation levels of and genes using quantitative methylation-specific PCR (qMSP). The relationship between the methylation levels of and genes and aging was analyzed using Spearman or Pearson correlation test. RESULTS: We found a significant positive correlation between the methylation levels of the two genes in these subjects ( < 0.05). In the overall population as well in the female subjects, methylation was found to be inversely correlated with age ( < 0.05). The methylation levels of and genes were inversely correlated with TG, ApoE, Lp(a) and AST in the overall population ( < 0.05). In both the female and male subjects, the methylation levels of the two genes were inversely correlated with Lp(a) ( < 0.05). In the male subjects, methylation was inversely correlated with AST ( < 0.05), while methylation was inversely correlated with HDL and ApoE ( < 0.05). In the female subjects, methylation was positively correlated with LDL and inversely correlated with ApoE and AST ( < 0.05). CONCLUSIONS The methylation levels of and are closely related to age and the levels of multiple proteins in healthy subjects.
Aging ; Cyclin-Dependent Kinase Inhibitor p15 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; DNA Methylation ; Female ; Humans ; Male ; Real-Time Polymerase Chain Reaction

OBJECTIVETo investigate the role of Tal1 gene, which is aberrantly expressed in 40%-60% of patients with T lymphocytic leukemia (T-ALL), in the proliferation of T-ALL cells.

METHODSWe established stable Jurkat-siTal1 and Jurkat-T1 cell lines by trasnfecting T-ALL Jurkat cells with lentiviral vectors to knock-down or overexpress Tal1. Jurkat cells transfected with negative control siRNAs for Tal1 knock-down (Jurkat-mock1) and over-expression(Jurkat-mock2) served as the control cells. The proliferation of the cells lines was assessed using CCK-8 assay, and the cell cycle distribution was determined by flow cytometry. The mRNA and protein expressions of cyclin-dependent kinase inhibitor 2 (CDKN2A) and cyclin-dependent kinase inhibitor 1 (CDKN2B) were measured by real-time RT-PCR and Western blotting, respectively.

RESULTSJurkat-T1 cells showed more active proliferation in vitro than Jurkat-mock2 cells, while Jurkat-siTal1 cells showed slower growth than Jurkat-mock1 cells. In Jurkat-T1 cells, G0/G1 phase cells were decreased and S phase cells increased compared with Jurkat-mock2 cells, and Jurkat-siTal1 cells showed increased G0/G1 phase cells and decreased S phase cells compared with Jurkat-mock1 cells. Real-time RT-PCR and Western blotting showed that Tal1 inhibited the cellular expression of CDKN2A and CDKN2B at both mRNA and protein levels.

CONCLUSIONTal1 promotes the growth and the transition from G0/G1 phase to S phase in T-ALL cells Jurkat by inhibiting the expressions of G0/G1 and S phase negative regulatory proteins CDKN2A and CDKN2B.

Apoptosis ; Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Cell Cycle ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p15 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Humans ; Jurkat Cells ; Lentivirus ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; metabolism ; pathology ; Proto-Oncogene Proteins ; metabolism ; RNA, Small Interfering ; T-Cell Acute Lymphocytic Leukemia Protein 1

OBJECTIVETo investigate the effect of monoamine oxidase inhibitor phenelzine on proliferation, apoptosis and histone modulation in acute lymphoblastic leukemia cell line Molt-4 cells.

METHODSThe effect of Phenelzine on cell proliferation was detected by MTT. Apoptotic rate was measured by flow cytometry. The variation of apoptosis associated proteins Caspase-3, Bcl-2 and Bax, cyclin-dependent kinase inhibitor p21, tumor suppressor protein p15, and the expression level of histone methylation of H3K4, H3K9 and histone acetylation of H3, DNMT1 were detected by Western Blot.

RESULTS① Molt-4 cell proliferation rates were (87.68±3.54)%, (67.84±3.24)%, (51.48±3.37)%, (28.72±2.56)% respectively after exposured to phenelzine at 5, 10, 15, 20 μmol/L for 24 h, P<0.05. ② After 10 μmol/L of phenelzine exposure for 24, 48, 72 h, cell proliferation rates were (67.84±3.24)%, (50.24±2.01)%, (40.31±2.25)%, P<0.05. ③ The apoptotic rates were (13.64±2.58)%, (31.24±3.42)%, (56.37±4.26)% after phenelzine treatment at 5, 10, 20 μmol/L for 24 h, which was concentration dependent. ④ Phenelzine could upregulate the expression of Bax, caspase-3, p21, and downregulate Bcl-2 expression. Phenelzine upregulated the methylation level of histone H3K4me1, H3K4me2 and histone acetylated H3, while it didn't change the level of histone H3K4me3, H3K9me1, H3K9me2. ⑤ Phenelzine inhibited DNMT1 expression and promoted p15 expression.

CONCLUSIONSPhenelzine increased the methylation of histone H3K4me1, H3K4me2, acetylation of histone H3 and p21, and decreased the expression of DNMT1 and p15, and ultimately inhibited the proliferation and apoptosis of Molt-4 cells.

Acetylation ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p15 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; metabolism ; Histones ; metabolism ; Humans ; Methylation ; Phenelzine ; pharmacology

OBJECTIVETo detect the presence of p15 antisense RNA(p15AS) in acute myeloid leukemia(AML).

METHODSp15AS and p15 mRNA in two leukemia cell lines was detected with strand-specific primer RT-qPCR. To explore the connection between p15AS and AML, 43 patients with newly diagnosed AML and 21 patients with benign diseases (Iron deficiency anemia) as controls were enrolled. The expression level of p15AS in bone marrow cells derived from the patients and the controls were determined by strand-specific primer RT-qPCR, and its relationship with clinical features was analyzed.

RESULTSThe two AML lines displayed high p15AS and low p15 expression. Samples derived from the AML patients showed relatively increased expression of p15AS and down-regulated p15 expression in their bone cells. In contrast, the 21 controls showed high expression of p15 but relatively low expression of the p15AS. Compared with the normal controls, the expression levels of p15 protein were significantly lower among the AML patients (P<0.01). No relationships were detected between the level of p15AS and the patient's age, gender, FAB subtype, total white blood cell count, platelet count, proliferative degree of bone marrow cell and karyotype classification (P>0.05 for all comparisons).

CONCLUSIONHigh expression of p15 antisense RNA was frequently found among AML patients, and may play an important role in epigenetic silencing of p15.

Adult ; Aged ; Bone Marrow Cells ; metabolism ; Cyclin-Dependent Kinase Inhibitor p15 ; genetics ; Epigenesis, Genetic ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; Male ; Middle Aged ; RNA, Antisense ; genetics ; metabolism ; Up-Regulation ; Young Adult

OBJECTIVETo explore the proliferation and invasive effects of inhibitors of kinase 4(INK4)(P15(ink4b) and P16(ink4a)/CDKN2) gene protein activation on RKO human colorectal cell in vivo and in vitro.

METHODSRKO human colorectal cell line was exposed to the specific DNA methyltransferase inhibitor 5-Aza-CdR and INK4(P15(ink4b) and P16(ink4a)/CDKN2) protein expression was detected by Western blotting. Soft agar cloning experiment and Transwell chamber assay were used to detect the proliferative and invasive ability in vitro. Tumorigenicity in nude mice was analyzed in vivo.

RESULTSINK4(P15(ink4b) and P16(ink4a)/CDKN2) protein expression of RKO human colorectal cells after exposure to 1×10(-7), 5×10(-7) and 1×10(-6) mol/L 5-Aza-CdR concentrations(A, B, C groups) were 1.13, 1.38, 1.92 folds and 1.11, 1.45, 2.14 folds compared to positive control group respectively. Soft agar cloning experiment showed the number of cell colony significantly decreased from 36.8±5.1(positive control group) to 32.4±7.2, 21.3±5.4 and 19.5±6.4 (3 experiment groups, all P<0.05) respectively. Transwell chamber assay showed that migrated cell number in positive control group(67.4±7.2) was significantly higher than those of 3 experimental groups(35.3±4.6, 29.5±7.3 and 25.3±6.2, respectively). The tumor volume of metastasis model in nude mice was inhibited in experimental groups, but not significantly lower compared to control group (P>0.05). There were significant differences of tumor weight and inhibition rate between control group and 3 experimental groups in nude mice respectively(all P<0.01).

CONCLUSIONINK4(P15(ink4b) and P16(ink4a)/CDKN2) protein activation can inhibit tumor proliferation, migration and suppress the tumor formation ability.

Animals ; Cell Line, Tumor ; Cell Proliferation ; Colorectal Neoplasms ; metabolism ; pathology ; Cyclin-Dependent Kinase Inhibitor p15 ; genetics ; metabolism ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; metabolism ; Humans ; Mice ; Mice, Inbred BALB C ; Neoplasm Metastasis ; Neoplasm Transplantation ; Transcriptional Activation

BACKGROUND: Aberrant DNA hypermethylation plays a pivotal role in carcinogenesis and disease progression; therefore, accurate measurement of differential gene methylation patterns among many genes is likely to reveal biomarkers for improved risk assessment. We evaluated the gene hypermethylation profiles of primary breast tumors and their corresponding normal tissues and investigated the association between major clinicopathological features and gene hypermethylation. METHODS: A single reaction using methylation-specific multiplex ligation-dependent probe amplification was used to analyze the DNA methylation status of 24 tumor suppressor genes in 60 cancerous tissues and their corresponding normal tissues from patients with primary breast cancer. RESULTS: In cancerous breast tissues, 21 of 24 genes displayed promoter methylation in one or more samples. The most frequently methylated genes included RASSF1 (43.3%), APC (31.7%), CDKN2B (25.0%), CDH13 (23.3%), GSTP1 (16.7%), and BRCA1 (10%). APC was associated with lymph node metastasis, and BRCA1 was associated with negative estrogen receptor and negative progesterone receptor expression. In normal breast tissues, 8 of 24 tumor suppressor genes displayed promoter hypermethylation; CDKN2B (28.3%) and RASSF1 (8.3%) hypermethylation were most frequently observed. CONCLUSIONS: RASSF1 and CDKN2B hypermethylation in Korean breast cancer patients were the most frequent in cancerous tissue and corresponding normal tissue, respectively. Our data indicates that methylation of specific genes is a frequent event in morphologically normal breast tissues adjacent to breast tumors as well as the corresponding breast cancers. This study also suggests that gene methylation is linked to various pathological features of breast cancer; however, this requires confirmation in a larger study.
Adult ; Breast/metabolism ; Breast Neoplasms/*genetics/metabolism/pathology ; Cyclin-Dependent Kinase Inhibitor p15/genetics ; *DNA Methylation ; Female ; Humans ; Lymphatic Metastasis ; Middle Aged ; Promoter Regions, Genetic ; Republic of Korea ; Tumor Suppressor Proteins/genetics

SUV39H1 is a histone 3 lysine 9 (H3K9)-specific methyltransferase that is important for heterochromatin formation and the regulation of gene expression. Chaetocin specifically inhibits SUV39H1, resulted in H3K9 methylation reduction as well as reactivation of silenced genes in cancer cells. Histone deacetylase (HDAC) inhibitors inhibit deacetylases and accumulate high levels of acetylation lead to cell cycle arrest and apoptosis. In this study, we demonstrated that treatment with chaetocin enhanced apoptosis in human leukemia HL60, KG1, Kasumi, K562, and THP1 cells. In addition, chaetocin induced the expression of cyclin-dependent kinase inhibitor 2B (p15), E-cadherin (CDH1) and frizzled family receptor 9 (FZD9) through depletion of SUV39H1 and reduced H3K9 methylation in their promoters. Co-treatment with chaetocin and HDAC inhibitor trichostatin A (TSA) dramatically increased apoptosis and produced greater activation of genes. Furthermore, this combined treatment significantly increased loss of SUV39H1 and reduced histone H3K9 trimethylation responses accompanied by increased acetylation. Importantly, co-treatment with chaetocin and TSA produced potent antileukemic effects in leukemia cells derived from patients. These in vitro findings suggest that combination therapy with SUV39H1 and HDAC inhibitors may be of potential value in the treatment of leukemia.
Acetylation/drug effects ; Adolescent ; Adult ; Aged ; Apoptosis/*drug effects ; Cadherins/metabolism ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p15/metabolism ; DNA Methylation/drug effects ; Enzyme Inhibitors/therapeutic use/*toxicity ; Frizzled Receptors/metabolism ; Gene Expression Regulation/drug effects ; HL-60 Cells ; Histone Deacetylase Inhibitors/therapeutic use/*toxicity ; Histone-Lysine N-Methyltransferase/*antagonists & inhibitors/metabolism ; Histones/genetics/metabolism ; Humans ; Hydroxamic Acids/therapeutic use/*toxicity ; K562 Cells ; Leukemia/drug therapy/metabolism/pathology ; Leukemia, Myeloid, Acute/genetics/metabolism/pathology ; Male ; Middle Aged ; Piperazines/therapeutic use/toxicity ; Promoter Regions, Genetic ; Young Adult

Previously, we found that high doses of genistein show an inhibitory effect on uterine leiomyoma (UtLM) cell proliferation. In this study, using microarray analysis and Ingenuity Pathways Analysis(TM), we identified genes (up- or down-regulated, > or = 1.5 fold, P < or = 0.001), functions and signaling pathways that were altered following treatment with an inhibitory concentration of genistein (50 microg/ml) in UtLM cells. Downregulation of TGF-beta signaling pathway genes, activin A, activin B, Smad3, TGF-beta2 and genes related to cell cycle regulation, with the exception of the upregulation of the CDK inhibitor P15, were identified and validated by real-time RT-PCR studies. Western blot analysis further demonstrated decreased protein expression of activin A and Smad3 in genistein-treated UtLM cells. Moreover, we found that activin A stimulated the growth of UtLM cells, and the inhibitory effect of genistein was partially abrogated in the presence of activin A. Overexpression of activin A and Smad3 were found in tissue samples of leiomyoma compared to matched myometrium, supporting the contribution of activin A and Smad3 in promoting the growth of UtLM cells. Taken together, these results suggest that down-regulation of activin A and Smad3, both members of the TGF-beta pathway, may offer a mechanistic explanation for the inhibitory effect of a high-dose of genistein on UtLM cells, and might be potential therapeutic targets for treatment of clinical cases of uterine leiomyomas.
Activins/*genetics/metabolism/pharmacology ; Anticarcinogenic Agents/*pharmacology ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cyclin-Dependent Kinase Inhibitor p15/genetics/metabolism ; Down-Regulation ; Female ; Genistein/*pharmacology ; Humans ; Leiomyoma/*metabolism ; Oligonucleotide Array Sequence Analysis ; Signal Transduction/drug effects ; Smad3 Protein/*genetics/metabolism ; Transforming Growth Factor beta/*genetics/metabolism ; Up-Regulation ; Uterine Neoplasms/*metabolism

OBJECTIVETo analyze the promoter methylation levels of p15, CDH1, DAPK and HICI genes of patients with myelodysplastic syndrome (MDS) and explore the relationship between the level of methylation and clinical features.

METHODSDNA methylation levels of p15, CDH1, DAPK and HICI in peripheral blood (PB) or bone marrow (BM) samples from 52 MDS patients were detected by real-time quantitative PCR. The correlation of the methylation level with clinical features and hematological findings was analyzed. 38 de novo AML patients and 46 normal individuals served as controls.

RESULTSThe methylation levels of p15, CDH1, DAPK and HICI were 16.23 ± 21.69, 6.59 ± 9.39, 0.14 ± 0.11 and 7.81 ± 9.70 in BM, and 14.96 ± 20.16, 6.00 ± 9.26, 0.12 ± 0.14 and 6.74 ± 9.72 in PB, respectively from 18 MDS patients, and the difference between BM and PB was not statistically significant (P > 0.05). The methylation levels of p15 (14.70 ± 18.17) and CDH1 (6.61 ± 8.79) genes in high risk (RAEBI/II) MDS were significantly higher than in low risk (RCMD/RARS/5q-, p15: 1.99 ± 1.59, CDH1: 1.23 ± 1.14 and RCMD, p15: 3.02 ± 3.42, CDH1:1.53 ± 2.06) MDS or control (p15: 1.69 ± 1.82, CDH1: 1.01 ± 1.12) (P < 0.05). The methylation levels of DAPK gene had no difference among subtypes of MDS, and that of HIC1 gene only differed between RAEB I/II (9.16 ± 11.95) and control (2.49 ± 2.26) (P = 0.042). The difference of methylation levels of p15, CDH1, DAPK and HICI in BM was statistically significant among subtypes of MDS (P = 0.001, 0.003, 0.039, 0.023, respectively). And so did of p15 and DAPK in PB (P = 0.013, 0.006, respectively). The methylation level of p15 and CDH1 was significantly correlated with IPSS classification and blasts percentage in BM.

CONCLUSIONSp15 and CDH1 genes are special hypermethylation genes in MDS. Methylation level of HIC1 gene showed an upward tendency from low risk to high risk MDS.

Adult ; Aged ; Aged, 80 and over ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Cadherins ; genetics ; metabolism ; Calcium-Calmodulin-Dependent Protein Kinases ; genetics ; metabolism ; Case-Control Studies ; Cyclin-Dependent Kinase Inhibitor p15 ; genetics ; metabolism ; DNA Methylation ; Death-Associated Protein Kinases ; Female ; Humans ; Kruppel-Like Transcription Factors ; genetics ; metabolism ; Male ; Middle Aged ; Myelodysplastic Syndromes ; genetics ; metabolism ; Promoter Regions, Genetic ; Real-Time Polymerase Chain Reaction ; Young Adult

OBJECTIVETo detect the expression and the CpG island methylation status of tumor suppressor gene p15 after exposure to 1,4-benzoquinone (1,4-BQ) in primary cultivated C57BL/6J mouse bone marrow cells in vitro.

METHODSThe mouse bone marrow cells were isolated in vitro. The effect of 0, 0.1, 1, 5, 10, 20, and 40 µmol/L 1,4-BQ on cell viability (CKK-8) was detected. 0, 0.1, 1, 10 µmol/L 1,4-BQ were used to intoxicate the mouse bone marrow cells for 24 h; Real-time PCR was employed to analyze the mRNA expression level of p15; The bisulfite sequencing PCR (BSP) was used to look into the methylation status of CpG islands in p15 promoter region.

RESULTS1,4-BQ exhibited dose-dependent toxicity to mouse bone marrow cells, and the LC(50) was 8.3 µmol/L (95%CI: 4.6 - 10.6 µmol/L). The mRNA expression of p15 in 10 µmol/L group was only equivalent to 43% of control group. Compared with control group, the decrease of p15 mRNA expression in1 and 10 µmol/L concentration were obvious, and the differences had statistical significance (P < 0.05 or P < 0.01). BSP sequencing results were same between the exposure groups and control group, the 56 CpG sites on CpG islands remained in the state of unmethylated.

CONCLUSIONmRNA expression of p15 gene decreases after exposure to 1,4-BQ, but the CpG islands methylation status in promoter is not affected, suggesting that methylation does not participate in 1,4-BQ-mediated p15 gene expression decrease, other effect mechanisms still need to be investigated.

Animals ; Base Sequence ; Benzoquinones ; toxicity ; Bone Marrow Cells ; metabolism ; Cells, Cultured ; CpG Islands ; Cyclin-Dependent Kinase Inhibitor p15 ; genetics ; DNA Methylation ; Environmental Exposure ; Mice ; Mice, Inbred C57BL ; Promoter Regions, Genetic ; RNA, Messenger ; genetics