|
| ||
|
|
||
Bisulfite DNA Sequencing (bisulfite + dna_sequencing)
Selected AbstractsDefective DNA methylation and CD70 overexpression in CD4+ T cells in MRL/lpr lupus-prone miceEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2007Abstract We have determined that abnormal DNA methylation in T cells coincides with the development of autoimmunity, using a mouse model that exhibits an age-dependent lupus-like disease (MRL/lpr mice). Splenic CD4+ T cells were isolated from these mice at 5 and 16,wk of age (before and after autoimmunity is established) and the expression of DNA methyltransferase,1 (Dnmt1) and the methylation-sensitive gene Tnfsf7 (CD70) was measured. Bisulfite DNA sequencing was used to monitor the methylation status of the Tnfsf7 gene. We found that Dnmt1 steady-state mRNA levels were significantly lower in 16-wk-old MRL/lpr mice, which had established autoimmunity, compared to the 5-wk-old MRL/lpr mice. Furthermore, the expression of CD70 was higher in MRL/lpr mice at 16,wk. CD70 was overexpressed in MRL/lpr mice compared to age- and sex-matched MRL+/+ controls. Bisulfite DNA sequencing of the Tnfsf7 gene in MRL/lpr mice revealed that at 16,wk, CG pairs were hypomethylated compared to 5-wk-old mice, and that Tnfsf7 from MRL/lpr mice was hypomethylated at 16,wk relative to age-matched MRL+/+ controls. Our data indicate that decreased expression of Dnmt1 and the corresponding T cell DNA hypomethylation correlate with the development of age-dependent autoimmunity in MRL/lpr mice. [source] DNA methylation and histone modifications cause silencing of Wnt antagonist gene in human renal cell carcinoma cell linesINTERNATIONAL JOURNAL OF CANCER, Issue 3 2008Ken Kawamoto Abstract Secreted frizzled-related protein 2 (sFRP2) is a negative modulator of the Wingless-type (Wnt) signaling pathway, and shown to be inactivated in renal cell carcinoma (RCC). However, the molecular mechanism of silencing of sFRP2 is not fully understood. Our study was designed to elucidate the silencing mechanism of sFRP2 in RCC. Expression of sFRP2 was examined in 20 pairs of primary cancers by immunohistochemistry. Kidney cell lines (HK-2, Caki-1, Caki-2, A-498 and ACHN) were analyzed for sFRP2 expression using real-time RT-PCR and Western blotting. The methylation status at 46 CpG sites of the 2 CpG islands in the sFRP2 promoter was characterized by bisulfite DNA sequencing. Histone modifications were assessed by chromatin immunoprecipitation (ChIP) assay using antibodies against AcH3, AcH4, H3K4 and H3K9. sFRP2 was frequently repressed in primary cancers and in RCC cells. The majority of sFRP2 negative cells had a methylated promoter. Meanwhile, sFRP2 expression was repressed by a hypomethylated promoter in Caki-1 cells, and these cells had a repressive histone modification at the promoter. In Caki-1 cells, sFRP2 was reactivated by trichostatin A (TSA). Repressive histone modifications were also observed in RCC cells with hypermethylated promoters, but sFRP2 was reactivated only by 5-aza-2,-deoxycytidine (DAC) and not by TSA. However, the activation of the silenced sFRP2 gene could be achieved in all cells using a combination of DAC and TSA. This is the first report indicating that aberrant DNA methylation and histone modifications work together to silence the sFRP2 gene in RCC cells. © 2008 Wiley-Liss, Inc. [source] Aberrant promoter methylation of insulin-like growth factor binding protein-3 gene in human cancersINTERNATIONAL JOURNAL OF CANCER, Issue 3 2007Kunitoshi Tomii Abstract Insulin-like growth factor binding protein-3 (IGFBP-3) is postulated to be a mediator of growth suppression signals. Here, we examined the methylation status of IGFBP-3 to correlate to clinicopathological factors in human cancers. The methylation status of IGFBP-3 was determined by bisulfite DNA sequencing and was correlated with expression semi-quantified by real-time RT-PCR to develop a methylation-specific PCR (MSP) assay for IGFBP-3. Using the MSP assay, we examined the methylation status of IGFBP-3 in gastric cancer (GC), colorectal cancer (CRC), breast cancer (BC) and malignant mesothelioma (MM). IGFBP-3 methylation was detected in 6 of 13 (46%) and 16 of 24 (67%) GC cell lines and tumors, respectively; 4 of 8 (50%) and 15 of 26 (58%) CRC cell lines and tumors, respectively; 3 of 11 (27%) and 7 of 39 (18%) BC cell lines and tumors, respectively and 1 of 5 (20%) and 18 of 56 (32%) MM cell lines and tumors, respectively. Interestingly, the methylation status of MM specimens from Japanese patients (75%, 12 out of 16 patients) was significantly higher than those from the USA (15%, 6 out of 40 patients) (p < 0.0001), suggesting the presence of ethnic differences in the IGFBP-3 methylation status. We also found that IGFBP-3 methylation was preferentially present in GCs arising in the lower-third of the stomach (p = 0.079). In summary, our results showed that IGFBP-3 methylation played an important role in the silencing of its expression, suggesting that IGFBP-3 may act as a tumor suppressor gene in several human cancers examined. © 2006 Wiley-Liss, Inc. [source] CpG methylation at promoter site ,140 inactivates TGF,2 receptor gene in prostate cancerCANCER, Issue 1 2005Hong Zhao M.D. Abstract BACKGROUND The action of transforming growth factor , (TGF-,) is mediated through type 1 (T,RI) and type 2 (T,RII) receptors. Prostate cancer cells are often resistant to TGF-, signaling due to loss of T,RII expression. The authors of the current study hypothesized that CpG methylation of the T,RII promoter at the Sp1 binding site ,140 mediates this loss of T,RII expression in prostate cancer. METHODS Sixty-seven prostate cancer (PC) samples, 8 benign prostatic hyperplasia (BPH) samples, and 4 prostate cancer cell lines (DUPro, LNCaP, ND-1 and PC-3) were analyzed for 1) T,RII mRNA expression by semiquantitative RT-PCR, 2) T,RII protein expression by immunohistochemistry, and 3) TGF,RII promoter methylation at CpG site ,140 by methylation specific PCR and bisulfite DNA sequencing. Prostate cancer cell lines were treated with the demethylating agent 5aza2,deoxycytidine to determine if T,RII gene expression could be increased by blocking promoter methylation. RESULTS mRNA and protein expression of T,RII was lower in the PC samples than in the BPH samples. CpG methylation at site ,140 was higher in PC than in BPH (P < 0.01). Promoter methylation was inversely correlated with T,RII mRNA expression in the PC and BPH samples (P < 0.0001). PC3, ND1, and DUPro T,RII mRNA expression increased following treatment of cells with 5-aza-2,-deoxycytidine. CONCLUSION CpG methylation of the T,RII promoter at CPG site ,140 leads to functional loss of the T,RII gene in prostate cancer. Treatment with 5-aza-2, deoxycytidine can restore gene expression. The current study results report the first association between prostate cancer and loss of the TGF- , signaling pathway by T,RII DNA promoter methylation. Cancer 2005;. © 2005 American Cancer Society. [source] | ||||||||||